An hbv rna sensing sequence, design method, nucleic acid construct and use

Abstract

The present patent application protects an HBV RNA sensing sequence, a design method, a nucleic acid construct and an application, wherein the HBV RNA sensing sequence is shown as any sequence of SEQ ID NO: 61-120. Based on the endogenous ADAR of cells, in the HBV replication cell model, by artificially designing a sensor for HBV RNA, the nucleic acid construct containing the HBV RNA sensing sequence is introduced into the host cell, which can specifically sense the HBV RNA transcript and produce downstream effects. When the downstream effect gene is replaced by a pro-apoptotic gene, it can theoretically specifically target HBV transcriptionally active cells and promote their apoptosis, suggesting that the use of this sensor for HBV RNA can be used for the development of new targeting strategies for HBV to eliminate cccDNA and integrated dslDNA, and promote the functional cure of slow hepatitis B.

Description

Technical Field

[0001] This invention belongs to the field of hepatitis B virus molecular biology, and relates to an HBV RNA sensing sequence, design method, nucleic acid construct and application, especially to a sensor for hepatitis B virus cccDNA and integrated DNA transcripts and its application. Background Technology

[0002] Chronic hepatitis B virus (HBV) infection is a major global public health problem, and infected individuals can develop cirrhosis and liver cancer. The key to persistent HBV infection lies in two unique molecular forms: persistent covalently closed circular DNA (cccDNA) in hepatocytes and double-stranded linear DNA (dslDNA) integrated into the host genome. cccDNA serves as a viral transcription template and persists in the nucleus of infected cells, while integrated dslDNA not only maintains viral protein expression but may also promote liver cancer development through insertional mutations.

[0003] While nucleoside (acid) analogs, currently the main drugs used clinically, can effectively inhibit viral replication, they cannot target and clear cccDNA and integrated viral genomes, leading to a high relapse rate after drug withdrawal. Novel targeted strategies, such as nucleic acid drugs and gene editing technologies, have shown potential in specifically clearing these molecular forms, but still suffer from significant drawbacks such as high relapse rates after drug withdrawal and off-target effects. Furthermore, the immune tolerance state caused by HBV infection also limits the effectiveness of immunotherapy. These problems mean that achieving clinical cure for chronic HBV infection remains a major challenge, necessitating the development of novel, targeted, and highly effective antiviral strategies.

[0004] During the host's innate immune response to viruses, the host often initiates downstream antiviral effects by sensing heterologous nucleic acid components such as viral RNA. However, this innate immune recognition is lacking during HBV infection due to various reasons. Therefore, artificially designed RNA sensors can be used to specifically sense HBV, and then applied to tracking or antiviral activities. By designing and constructing artificially targeted RNA sequences, endogenous RNA editing enzymes can be recruited via the sensing target RNA to perform site-specific editing of the delivered nucleic acid, regulating specific gene expression and producing effects. ADAR (Adenosine Deaminases Acting on RNA), as a type of RNA adenosine deaminase, can specifically convert adenosine (A) to inosine (I), which is recognized as guanosine (G) during translation and plays an important role in the host's innate immune response. Therefore, ADAR-mediated RNA editing can achieve targeted editing of specific RNA sequences. Theoretically, artificial receptors based on this endogenous ADAR possess high specificity and low off-target risk. Therefore, they can specifically sense viral RNA-positive cells, and when downstream effector genes are designed as pro-apoptotic genes, they can be used to specifically target HBV-transcribed cells and promote apoptosis, thereby clearing cccDNA and integrated dslDNA, providing technical support for the functional cure of chronic hepatitis B. However, there are currently no studies or reports on the design of artificial RNA receptors based on endogenous ADAR to specifically sense HBV RNA-positive cells. Summary of the Invention

[0005] The purpose of this invention is to address at least one deficiency in existing technologies, such as the inability of current clinical and investigational methods to target and eradicate HBV-positive cells. This invention provides an HBV RNA sensing sequence, a design method, a nucleic acid construct, and its applications. Based on endogenous RNA deaminases, this invention designs a sensing sequence targeting common regions of HBV covalently closed circular DNA (cccDNA) and double-stranded linear DNA (dslDNA) transcripts, developing a sensor for HBV RNA. Introducing a nucleic acid construct containing the HBV RNA sensing sequence into host cells allows for the specific sensing of HBV RNA transcription and the generation of downstream effects. This suggests that this HBV RNA sensor could be used to develop novel targeting strategies for HBV to promote the clearance of cccDNA and integrated dslDNA, thereby promoting functional cure of chronic hepatitis B.

[0006] This invention provides a sensor reporter capable of recognizing hepatitis B virus (HBV) RNA molecules. This RNA sensor reporter is designed targeting sequences of common transcripts of HBV cccDNA and HBV dslDNA. Upon binding to the target RNA, this sequence activates the expression of downstream reporter effector genes based on endogenous RNA deaminases, achieving specific sensing, reporting, and response to HBV RNA transcripts. The sequences targeting the common transcripts of HBV cccDNA and HBV dslDNA are shown in SEQ ID No. 1 to SEQ ID No. 60. The sequence and design of this HBV sensor can be applied to research on the HBV life cycle, advancing the understanding of viral transcriptional characteristics and the development of new targeting strategies.

[0007] In this patent, an HBV RNA sensor refers to a nucleic acid construct that contains a HBV RNA sensing sequence and can produce a specific effect after sensing HBV target RNA.

[0008] To address the aforementioned technical problems, the first aspect of this invention provides a method for designing HBV RNA sensing sequences, comprising the following steps:

[0009] The designed HBV RNA sensing sequence is 200-350 nucleotides in length;

[0010] The designed HBV RNA sensing sequence and the reverse complementary sequence of the target RNA have more than 80% homology.

[0011] Multiple symmetrically spaced hairpin loop structures were inserted into the HBV RNA sensing sequence;

[0012] Point mutations were performed on the in-frame stop codon and start codon of the HBV RNA sensing sequence;

[0013] The target RNA is a shared sequence of HBV cccDNA and dslDNA transcripts.

[0014] In a preferred embodiment, the length of the designed HBV RNA sensing sequence is 200-350 nucleotides, preferably including: the length of the designed HBV RNA sensing sequence is 220-300 nucleotides; more preferably including 220-280 nucleotides; even more preferably including 240-270 nucleotides; for example, 252 nucleotides.

[0015] In a preferred embodiment, the HBV RNA sensing sequence is designed to have more than 80% homology with the reverse complementary sequence of the target RNA, preferably including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% homology.

[0016] In a preferred embodiment, the HBV RNA sensing sequence is designed to have 80%–90% homology with the reverse complementary sequence of the target RNA.

[0017] In a preferred embodiment, the HBV RNA sensing sequence is designed to have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homology with the reverse complementary sequence of the target RNA.

[0018] In a preferred embodiment, the design method involves point mutation of the in-frame stop codon and start codon of the HBV RNA sensing sequence, wherein the stop codon is TAG, TGA, or TAA, and the start codon is ATG.

[0019] In a preferred embodiment, the point mutations include: TAG mutation to TAC; TGA mutation to TGC; TAA mutation to TAC; ATG mutation to ATC;

[0020] As understood by those skilled in the art, the aforementioned homology refers to the percentage of the number of bases in the sensing sequence that are complementary to the target RNA in the length of the target RNA sequence.

[0021] As understood by those skilled in the art, the HBV RNA refers to the RNA sequence of chronic hepatitis B virus (HBV).

[0022] As understood by those skilled in the art, the HBV cccDNA refers to covalently closed circular DNA (cccDNA) in HBV.

[0023] As understood by those skilled in the art, the dslDNA refers to double-stranded linear DNA (dslDNA) integrated into the host genome by HBV.

[0024] In a preferred embodiment, the length of the shared sequence is 150-200 bp.

[0025] In a specific implementation, the common sequence is as shown in any sequence of SEQ ID NO:1 to 60.

[0026] In one specific implementation, the HBV RNA sensing sequence includes a core sequence that is complementary to nucleotides 65-115 of the common sequence and has 1-5 base mismatches.

[0027] In one specific implementation, the core sequence is complementary to nucleotides 65-115 of the common sequence and there are 1, 2, 3, 4 or 5 base mismatches.

[0028] In a preferred embodiment, the mismatch includes pairing the common sequence 5'CCA with the core sequence 5'TAG.

[0029] In a preferred embodiment, the mismatch further includes mismatches formed by point mutations in the stop codon or start codon within the frame, as described above.

[0030] In a preferred embodiment, the common sequence and the HBV RNA sensing sequence have 1-10 mismatch sites.

[0031] In a preferred embodiment, there are four symmetrically spaced hairpin ring structures, located on the flanks of the core sequence.

[0032] In one specific embodiment, the sequences of the hairpin ring structures are shown as SEQ ID NO:121 to 124, respectively.

[0033] The sequence corresponding to SEQ ID NO:121 is: agacatgaggatcacccatgt.

[0034] The sequence corresponding to SEQ ID NO:122 is: aagggtggaggaacaccccaccct.

[0035] The sequence corresponding to SEQ ID NO:123 is: acagaagcaccatcagggcttctg.

[0036] The sequence corresponding to SEQ ID NO:124 is: gtgcgtggagcatcagcccacgca.

[0037] In a specific implementation scheme, the HBV RNA sensing sequence obtained by design is shown as any sequence of SEQ ID NO:61~120.

[0038] A second aspect of the present invention provides an HBV RNA sensing sequence, wherein the HBV RNA sensing sequence is 200-350 nucleotides in length; the HBV RNA sensing sequence has 80%-90% homology with the reverse complementary sequence of the target RNA; the HBV RNA sensing sequence contains multiple symmetrically spaced hairpin loop structures; the in-frame stop codon and start codon of the HBV RNA sensing sequence contain point mutations; wherein the target RNA is a common sequence of HBV cccDNA and dslDNA transcripts.

[0039] The sensing sequence is defined in this patent as a sequence that, after transcription, can fully or partially pair complementary with the HBV target RNA sequence and recruit endogenous ADAR to perform "A to I" editing on it.

[0040] In a preferred embodiment, the length of the HBV RNA sensing sequence is as described in the design method of the first aspect of the present invention.

[0041] In a preferred embodiment, the homology between the HBV RNA sensing sequence and the reverse complementary sequence of the target RNA is as described in the design method of the first aspect of the present invention.

[0042] In a preferred embodiment, the point mutation is as described in the design method of the first aspect of the present invention.

[0043] In a preferred embodiment, the stop codon and the start codon are as described in the design method of the first aspect of the invention.

[0044] In one specific implementation, the HBV RNA sensing sequence is as shown in any sequence of SEQ ID NO:61-120.

[0045] In one specific implementation, the HBV RNA sensing sequence is obtained by the design method described in the first aspect of the present invention.

[0046] In a preferred embodiment, the HBV RNA sensing sequence can specifically target transcripts of the common region of HBV cccDNA and dslDNA.

[0047] A third aspect of the present invention provides a nucleic acid construct comprising, from the 5' end to the 3' end, the following sequences in sequence: (i) a promoter sequence, (ii) a spacer sequence, (iii) a linker sequence, (iv) an HBV RNA sensing sequence as described in the second aspect of the present invention, (v) a linker sequence, (vi) an effector gene sequence, and (vii) a polyA sequence.

[0048] In a preferred embodiment, after the nucleic acid construct transcribes a transcript containing the HBV sensing sequence, it can partially complement the target RNA. The AC mismatch site can recruit endogenous ADAR to edit A into hypoxanthine (I). I is recognized by the host ribosome as G, thereby invalidating the stop codon and allowing downstream codons to be read through, thus producing the corresponding downstream effects.

[0049] In a preferred embodiment, the promoter sequence in the nucleic acid construct corresponds to either the polymerase II (polII) promoter or the polymerase III (polIII) promoter.

[0050] In a preferred embodiment, the HBV sensing sequence of the nucleic acid construct is designed in a conserved region of the HBV gene, and can target HBV genotypes A to H.

[0051] In a preferred embodiment, the polymerase II (pol II) promoter includes the CMV promoter.

[0052] In a preferred embodiment, the polymerase III (pol III) promoter includes the U6 promoter.

[0053] In a preferred embodiment, the spacer sequence in the nucleic acid construct is 700-1000 nucleotides in length.

[0054] In a preferred embodiment, the spacer sequence is 800, 850, 900, 950, or 1000 nucleotides in length.

[0055] In a preferred embodiment, the spacer sequence in the nucleic acid construct is 700-900 nucleotides in length, for example, 720-800 nucleotides.

[0056] In a preferred embodiment, the effector gene sequence in the nucleic acid construct corresponds to a fluorescent reporter gene.

[0057] In a preferred embodiment, the linker sequence in the nucleic acid construct is a self-cleaving 2A peptide;

[0058] In a preferred embodiment, the self-cleaving 2A peptide is selected from any one of P2A peptide, T2A peptide, F2A peptide, and E2A peptide.

[0059] In a preferred embodiment, the nucleic acid construct can be transcribed to an mRNA sequence that is at least partially complementary to the target RNA.

[0060] The nucleic acid construct described in this invention can recruit endogenous RNA adenosine deaminase (ADAR).

[0061] The fourth aspect of the present invention also provides the use of the sensing sequence as described in the second aspect of the present invention or the nucleic acid construct as described in the third aspect of the present invention in the preparation of a medicament for treating HBV infection.

[0062] In addition, other aspects of the present invention provide a recombinant plasmid comprising the nucleic acid construct as described in the second aspect of the present invention.

[0063] In a preferred embodiment, the plasmid backbone is pcDNA3.1.

[0064] In addition, other aspects of the present invention provide a recombinant cell, which includes the formation of a cell by introducing a recombinant plasmid as described above into a cell, said cell being a human embryonic kidney cell or a liver cancer cell.

[0065] In a preferred embodiment, the cells are HEK-293T cells or Huh7 cells.

[0066] Other aspects of the present invention also provide the use of recombinant plasmids as described above or recombinant cells as described above in the preparation of medicaments for treating HBV infection.

[0067] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0068] This invention, based on the endogenous RNA editing mechanism within cells, utilizes an artificially designed sensor targeting HBV RNA in an HBV replication cell model. A nucleic acid construct containing HBV RNA sensing sequences is introduced into host cells, specifically sensing HBV RNA transcription and generating downstream effects through RNA editing. When the downstream effector gene is replaced with a pro-apoptotic gene, theoretically, it can specifically target cells with active HBV transcription and promote apoptosis. This suggests that this HBV RNA-targeting sensor could be used to develop novel HBV targeting strategies to clear cccDNA and integrated dslDNA, promoting functional cure of chronic hepatitis B. Attached Figure Description

[0069] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings:

[0070] Figure 1 This is a schematic diagram illustrating the design and working principle of a nucleic acid construct targeting HBV RNA sensor in one embodiment of the present invention.

[0071] Figure 2This is a schematic diagram showing the results of fluorescence microscopy observation and Western Blot detection of downstream effector gene expression after co-transfection of HEK-293T cells with the nucleic acid construct targeting HBV RNA and pHBV1.3 plasmid in one embodiment of the present invention.

[0072] Figure 3 This is a schematic diagram showing the results of fluorescence microscopy observation and Western Blot detection of downstream effector gene expression after co-transfection of Huh7 cells with the nucleic acid construct targeting HBV RNA and pHBV1.3 plasmid in one embodiment of the present invention.

[0073] Figure 4 This is a schematic diagram showing the results of flow cytometry analysis of the proportion of HEK-293T cells (GFP+ cells) with positive effector gene expression in one embodiment of the present invention.

[0074] Figure 5 This is a schematic diagram showing the results of flow cytometry analysis of the proportion of Huh7 cells (GFP+ cells) with positive effector gene expression in one embodiment of the present invention.

[0075] Figure 6 This is a schematic diagram showing the results of detecting the expression levels of total HBV RNA and the mRNA in the HBV sensing sequence target region using qPCR in one embodiment of the present invention. Detailed Implementation

[0076] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0077] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0078] In the following examples, the experimental materials involved include: HEK-293T cells and Huh7 cells, both of which were long-term stored in the laboratory. The pHBV1.3 plasmid, which is 1.3 times the length of the HBV genome, was also long-term stored in the laboratory.

[0079] In the following embodiments, the steps for analyzing probe hybridization and detection results include:

[0080] (1) Immunoblotting experiment: Protein samples were collected using lysis buffer containing protease inhibitors and phosphatase inhibitors, 5× loading buffer was added, and the mixture was heated at 100℃ for 10 min. Proteins were separated by 8% SDS-PAGE gel electrophoresis at 120V (protein loading volume was 10 μL), and then the proteins were transferred to an NC membrane. The membrane was blocked with 5% skim milk powder at room temperature for 1 h, and then incubated overnight with the corresponding antibody. The NC membrane incubated with the primary antibody was washed with PBST, and the corresponding secondary antibody was added and incubated for 1 h. After PBST, ECL chemiluminescence reagent was added and reacted for 2 min. The membrane was then exposed in a dark room, and the results were analyzed and photographed.

[0081] (2) Flow cytometry analysis of the percentage of positive cells: Transfected cells were digested with trypsin to prepare a single-cell suspension, and the concentration was adjusted to 1-5 × 10⁻⁵. 6 / ml and resuspend in PBS. Before transfection, remove cell clumps through a 100μm filter, and prepare untransfected cells as a negative control. During transfection, first adjust the voltage using the negative control to ensure it is in the appropriate position, then collect a sufficient number of cells. For data analysis, delineate the main cell population, remove debris, set the fluorescence positivity threshold based on the negative control, and calculate the percentage of positive cells.

[0082] (3) RT-qPCR experiment: Trizol was used to lyse transfected cells and reverse transcription and real-time quantitative PCR were performed. GAPDH was used as an internal reference gene and the relative expression level of mRNA was calculated according to the 2^(-△△Ct) method.

[0083] Example 1 - Design and Vector Construction of HBV RNA Sensing Sequence

[0084] This embodiment is based on the deamination RNA editing function of endogenous ADAR, and designs an HBV RNA sensing sequence that targets the common region of HBV cccDNA and dslDNA transcripts (see principle). Figure 1 The specific steps include:

[0085] (1) Analyze the conserved regions of each HBV genotype. Using the HBV genotype D sequence as a reference, and taking into account genotypes A, B, and C, design the sequence according to the following method. The specific steps are as follows:

[0086] The designed HBV RNA sensing sequence is 200-350 nucleotides in length;

[0087] The designed HBV RNA sensing sequence has more than 80% homology with the reverse complementary sequence of the target RNA (the common sequence of HBV cccDNA and dslDNA transcripts).

[0088] Multiple symmetrically spaced hairpin loop structures were inserted into the HBV RNA sensing sequence;

[0089] Point mutations were performed on the in-frame stop codon and start codon of the HBV RNA sensing sequence.

[0090] In addition, all target sequences that can be used for sensing were screened from the common positive strand sequences of HBV cccDNA and dslDNA, as shown in the table below:

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] (2) HBV RNA sensing sequences were designed for the target sequences described above. These sequences were 200-350 nucleotides in length, including a 51-nucleotide core sequence containing a 5' TAG sequence that mismatches the 5'CCA of the target RNA sequence. Two hairpin loops were designed on either side of the core sequence to reduce RNA folding and slow ribosome movement, preventing the stop codon from being read. Furthermore, point mutations were performed on the in-frame stop codons TAG, TGA, and TAA, and the in-frame start codon ATG in the HBV RNA sensing sequences to remove their additional stop or start codon functions. The HBV RNA sensing sequences are shown in the table below (uppercase sequences are those matching the target sequence, lowercase sequences are hairpin loop sequences and point mutation sites):

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] (3) Select two HBV RNA sensing sequences, sesHBV49 and sesHBV47, and add CMV promoter, mCherry reporter gene and P2A peptide upstream of the sequence, and add T2A peptide, eGFP reporter gene and bGH polyA sequence downstream. Then link them to the pcDNA3.1 backbone and label them as F1 and F2, respectively.

[0107] Example 2 - Detection of receptor effects in an HBV replicating cell model

[0108] This embodiment uses the HBV RNA receptors F1 and F2 constructed in Example 1 to detect their sensing effect on HBV RNA. Specific steps include:

[0109] (1) Preparation of cell samples;

[0110] HEK-293T cells or Huh7 cells were administered at a rate of 0.5 × 10⁻⁶. 6 Cells were seeded into 24-well plates and allowed to grow to approximately 80% confluence. Plasmids were then transfected at a rate of 0.5 μg / well. After 48 hours of transfection, the cells were observed and photographed under a fluorescence microscope. Cell samples were collected and Western blot was used to detect eGFP expression. The percentage of positive cells was determined by flow cytometry.

[0111] (2) Experimental results;

[0112] like Figure 2 and Figure 3 As shown, compared to the control group, significant eGFP fluorescence signals were observed in groups simultaneously transfected with F1 and pHBV1.3 or simultaneously transfected with F2 and pHBV1.3, and Western blotting detected significant eGFP expression. Figure 4 and Figure 5 As shown, flow cytometry analysis also detected an increase in the proportion of cells that were both mCherry and eGFP positive.

[0113] The results above indicate that in HEK-293T cells and Huh7 cells, receptors targeting the common region of HBV cccDNA and dslDNA transcripts can sense HBV RNA through activation of the downstream eGFP reporter gene.

[0114] Example 3 - Effects of receptors on target gene transcription

[0115] This embodiment verifies the effect of receptors F1 and F2 designed in Example 1 on target gene transcription, specifically including the following steps:

[0116] (1) Experimental procedure:

[0117] Cell samples collected from transfection in Example 1 were lysed with 500 μL of trizol and collected in an EP tube. 100 μL of chloroform was added, mixed, and allowed to stand at room temperature for 5 min. The tube was then centrifuged at 12000 g for 15 min at 4 °C. The supernatant was transferred to a new EP tube, and an equal volume of isopropanol was added. The tube was incubated at -20 °C overnight. The tube was centrifuged at 12000 g for 15 min at 4 °C, and the supernatant was discarded. 1 ml of 75% DEPC ethanol was added, and the tube was centrifuged at 12000 g for 5 min at 4 °C, and the supernatant was discarded. After air-drying, 20 μL of ddH2O was added to dissolve the RNA. The AdvanceFast 1st StrandcDNA Synthesis Kit reverses RNA into cDNA, utilizing... qPCR was performed using SYBR Green Master Mix for real-time quantitative PCR. Primers targeting HBV total RNA and HBV RNA sensing sequence target regions were designed for detection. GAPDH was used as an internal reference gene, and the relative expression level of mRNA was calculated using the 2^(-ΔΔCt) method.

[0118] The primer sequences are as follows:

[0119] sesHBV1-F: 5'-AGACTTGGCCCCCAATACCA-3' (SEQ ID NO: 125);

[0120] sesHBV1-R: 5'-TTACTAGTGCCCAGAAGCCC-3' (SEQ ID NO: 126);

[0121] sesHBV2-F: 5'-CACTCCAAGGGTGGAGGAAC-3' (SEQ ID NO: 127);

[0122] sesHBV2-R: 5'-TGGATGACTACTGCGCAAGG-3' (SEQ ID NO: 128);

[0123] GAPDH: F-5'-GGTATCGTGGAAGGACTCATGA-3' (SEQ ID NO: 129); R-5'-ATGCCAGTGGCTTC CCGTTCAGC-3' (SEQ ID NO: 130).

[0124] (2) Experimental Results

[0125] like Figure 6 As shown, the receptors F1 and F2 targeting HBV RNA did not significantly affect the transcription of the target gene.

[0126] As can be seen from the above embodiments, the present invention is designed to target HBV RNA receptors derived from the common region of HBV cccDNA and dslDNA, which can generate expression of downstream specific genes in cells that sense active HBV transcription, without significantly affecting the transcription of target genes, thus advancing the understanding of the life cycle of hepatitis B virus and the development of new targeting strategies.

[0127] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for designing HBV RNA sensing sequences, comprising the following steps: The designed HBV RNA sensing sequence is 200-350 nucleotides in length; The designed HBV RNA sensing sequence and the reverse complementary sequence of the target RNA have more than 80% homology. Multiple symmetrically spaced hairpin loop structures were inserted into the HBV RNA sensing sequence; Point mutations were performed on the in-frame stop codon and start codon of the HBV RNA sensing sequence; The target RNA is a shared sequence of HBV cccDNA and dslDNA transcripts.

2. The design method as described in claim 1, characterized in that, The length of the shared sequence is 150-200 bp; preferably, the shared sequence is any sequence of SEQ ID NO:1 to 60.

3. The design method as described in claim 1 or 2, characterized in that, The HBV RNA sensing sequence is designed to include a core sequence that is complementary to nucleotides 65-115 of the common sequence and has 1-5 base mismatches; preferably, the mismatches include the pairing of 5'CCA of the common sequence with 5'TAG of the core sequence.

4. The design method according to any one of claims 1-3, characterized in that, The number of the symmetrically spaced hairpin loop structures is four, which are located on the flanks of the core sequence; preferably, the sequences of the hairpin loop structures are as shown in SEQ ID NO: 121 to 124.

5. The design method according to any one of claims 1-3, characterized in that, The HBV RNA sensing sequences obtained by the design are shown as any sequence in SEQ ID NO:61~120.

6. An HBV RNA sensing sequence, characterized in that, Obtained by the design method described in any one of claims 1-5.

7. A nucleic acid construct comprising, from the 5' end to the 3' end, the following sequences: (i) a promoter sequence, (ii) a spacer sequence, (iii) a linker sequence, (iv) the HBV RNA sensing sequence as described in claim 6, (v) a linker sequence, (vi) an effector gene sequence, and (vii) a polyA sequence.

8. The nucleic acid construct as described in claim 7, characterized in that, The nucleic acid construct meets one or more of the following conditions: The promoter sequence corresponds to either the polymerase II (pol II) promoter or the polymerase III (pol III) promoter; The spacer sequence is 700-1000 nucleotides in length; And / or, the effector gene sequence corresponds to a fluorescent reporter gene.

9. The nucleic acid construct as described in claim 7 or 8, characterized in that, The linker sequence is a self-cleaving 2A peptide, preferably selected from any one of P2A peptide, T2A peptide, F2A peptide, and E2A peptide.

10. The use of the sensing sequence as described in claim 6 or the nucleic acid construct as described in any one of claims 7-9 in the preparation of a medicament for treating HBV infection.

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