A cyclic triplex forming oligonucleotide, a preparation method and application thereof in preparing tumor targeting drugs

Abstract

The application relates to a circular triplex-forming oligonucleotide, a preparation method and application in preparation of tumor-targeting drugs. The circular triplex-forming oligonucleotide (Cir-TFO) is composed of two oligonucleotide chains A and B, each of which comprises a first complementary sequence, a first connecting sequence, a target sequence, a second connecting sequence and a second complementary sequence from the 5' end to the 3' end. The first complementary sequences of the two oligonucleotide chains A and B are reversely and complementarily connected, and the second complementary sequences are reversely and complementarily connected to form a closed ring structure. The target sequence is a TFO sequence capable of forming a triplex structure with double-stranded DNA in the promoter region of a target gene through Hoogsteen hydrogen bonds, and the target gene is selected from a proto-oncogene, an anti-apoptotic gene or a tumor metabolism-related gene.

Description

Technical Field

[0001] This invention relates to the field of nucleotides, and in particular to the preparation and application of artificial cyclic triple-stranded oligonucleotides in nucleic acid technology. Background Technology

[0002] Traditional cancer treatments, such as chemotherapy and radiotherapy, while capable of inhibiting tumor growth to some extent, often come with severe side effects. In recent years, gene therapy has garnered significant attention due to its high targeting and fewer side effects. Triplex-forming oligonucleotides (TFOs) are a class of oligonucleotides that can specifically bind to double-stranded DNA via Hoogsteen hydrogen bonds to form a triple-stranded structure. They can induce DNA damage and suppress oncogene expression by binding to specific sequences in the genome. In cancer treatment, TFO sequences have the potential to inhibit tumor growth, induce tumor cell apoptosis, or enhance the sensitivity of tumor cells to other treatments by regulating the expression of tumor-related genes.

[0003] Current TFO design research largely focuses on the binding of single-stranded oligonucleotides to target DNA. However, single-stranded nucleotide chains suffer from poor intracellular stability, making it difficult to maintain stable and sustained therapeutic effects in tumor-targeted therapy. Therefore, improving the structure of TFOs to enhance their intracellular stability and thus improve their efficacy in tumor-targeted therapy has become a pressing issue that needs to be addressed in current technologies. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, this invention provides a Circular Triplex-Forming Oligonucleotide (Cir-TFO) structure and its preparation method, and explores its targeted application in tumor therapy. Specifically, the technical solution provided by this invention is as follows:

[0005] A circular triple-stranded oligonucleotide (Cir-TFO) consists of two oligonucleotide chains, A and B. Each oligonucleotide chain includes, from its 5' end to its 3' end, a first complementary sequence, a first linker sequence, a target sequence, a second linker sequence, and a second complementary sequence. The first complementary sequences of the two oligonucleotide chains A and B are anticomplementary and linked together, forming a closed circular structure. The target sequence is a TFO sequence capable of forming a triple-stranded structure with double-stranded DNA in the promoter region of a target gene via Hoogsteen hydrogen bonds. The target gene is selected from proto-oncogenes, anti-apoptotic genes, or tumor metabolism-related genes.

[0006] The aforementioned cyclic triple-stranded oligonucleotide further includes, but is not limited to, c-MYC, BCL2, KRAS, EGFR, or VEGF as the target gene; the TFO sequence is 8-30 bases in length and targets the purine-rich or pyrimidine-rich chain in the promoter region of the target gene.

[0007] In the aforementioned cyclic triple-stranded oligonucleotide, the lengths of the first and second complementary sequences are 6-20 bases, and the lengths of the first and second linker sequences are 4-8 bases.

[0008] The circular triple-stranded oligonucleotide further specifies that the target sequence, as shown in SEQ ID NO:1, specifically targets the purine-rich strand of double-stranded DNA located 40 bp upstream of the MYC gene P2 promoter at an 11 bp position. The target sequence binds to the purine-rich strand of the double-stranded DNA via Hoogsteen hydrogen bonds, forming a triple-stranded structure (Triplex DNA), thereby inhibiting the transcriptional activation of the MYC gene. The first and second complementary sequences of strand A are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively. The first linker sequence of both strands A and B is AGTT, and the second linker sequence of both strands A and B is TGAT.

[0009] This invention also provides a method for preparing any one of the cyclic triple-stranded oligonucleotides, comprising the following steps:

[0010] a) Synthesize oligonucleotide chain A and oligonucleotide chain B respectively;

[0011] b) Two oligonucleotide chains, A and B, are annealed or enzymatically linked in a cyclization buffer to form a closed cyclic structure.

[0012] c) Purify the product to obtain the final product.

[0013] The preparation method described herein further includes:

[0014] The cyclization buffer contains 10-100 mM Tris-HCl (pH 7.0-8.5), 1-10 mM EDTA, 20-200 mM NaCl, and 5-20% glycerol; the annealing reaction program is as follows: denaturation at 80-95℃ for 1-10 minutes, cooling to 20-40℃ at a rate of 0.5-2℃ / cycle, for a total of 10-100 cycles.

[0015] The present invention also provides a pharmaceutical composition comprising any of the cyclic triple-stranded oligonucleotides and a pharmaceutically acceptable carrier.

[0016] The pharmaceutical composition further includes a carrier selected from liposomes, exosomes, polymer nanoparticles, or viral vectors, and the pharmaceutical composition is intended for injection, oral administration, or topical delivery.

[0017] This invention also provides the application of any of the aforementioned cyclic triple-strand oligonucleotides in the preparation of tumor-targeting drugs, which, by targeting and binding to the promoter region of the aforementioned genes, induce DNA double-strand breaks (DSBs), inhibit transcriptional activation, or silence epigenetic information, thereby inhibiting tumor cell proliferation, migration, or inducing apoptosis. The tumor is a malignant tumor, including solid tumors and non-solid tumors.

[0018] The beneficial effects of this invention are as follows: Compared with the prior art, the cyclic triple-stranded oligonucleotide provided by this invention eliminates the free ends of linear oligonucleotides through a closed cyclic structure. Utilizing the steric hindrance effect of complementary cyclic sequences, the cyclic TFO structure has no free ends, preventing exonucleases from attacking the TFO sequence from the ends. Furthermore, the steric hindrance generated by its own structure hinders the binding and interaction of TFO with nucleases, allowing it to maintain activity in cells for a longer period and enabling direct use in tumor-targeted therapy. In terms of preparation process, this invention promotes cyclization through complementary regions of specific sequences, improving reaction efficiency. Employing a double-stranded complementary-driven cyclization strategy and a gradient annealing procedure, it eliminates the need for enzymes or cross-linking agents, effectively reducing process costs and demonstrating good economic efficiency and feasibility. From a therapeutic perspective, the cyclic TFO provided by this invention exhibits high efficiency and safety, with advantages such as high stability, ease of preparation and transfection. It also shows therapeutic potential against various tumors, possessing broad-spectrum therapeutic advantages, and providing a novel and highly valuable solution for the field of tumor treatment. Attached Figure Description

[0019] Figure 1 A schematic diagram showing the target sequence location of the MYC gene used in the preparation of circular TFO in Example 1.

[0020] Figure 2 This is a gel electrophoresis image of the cyclic TFO product prepared in Example 1.

[0021] Figure 3 This is a comparison chart showing the results of the Cell Counting Kit 8 (CCK-8) assay in Example 2, used to determine the ability of Cir-TFO to inhibit the proliferation of colorectal cancer cells.

[0022] Figure 4 The graphs show the effects of Cir-TFO on the migration and colony formation inhibition of malignant tumor cells in Example 2.

[0023] Figure 5 This is a graph showing the change in tumor volume with drug administration time in the animal experiment of Example 2.

[0024] Figure 6 The images show H&E staining of the major organs (heart, liver, spleen, lung, and kidney) of the tumor-bearing mice in each group after drug administration in Example 2. Detailed Implementation

[0025] The present invention will be further described below with reference to the embodiments. However, the present invention is not limited to the following embodiments. Similar procedures using Cir-TFO to target any gene or treat any type of tumor cells should fall within the scope of this patent method.

[0026] Example 1: Preparation method of cyclic TFO (Cir-TFO)

[0027] 1. Synthesize oligonucleotide chains A and B, respectively, as linear precursor DNA.

[0028] The linear precursor DNA was synthesized by Tsingke Biotechnology Co., Ltd. using the standard phosphoramidite-triester method. During synthesis, according to the required base sequence, the corresponding phosphoramidite monomers were sequentially coupled to a solid-phase support. After deprotection, oxidation, and capping steps, linear single-stranded DNA was finally obtained, which was purified by polyacrylamide gel electrophoresis (PAGE) and verified by mass spectrometry (MS). The synthesized DNA was delivered as a lyophilized powder and stored at -20°C for later use.

[0029] Each oligonucleotide chain, from its 5' end to its 3' end, includes a first complementary sequence, a first linker sequence, a target sequence, a second linker sequence, and a second complementary sequence, in sequence. The first complementary sequences of oligonucleotide chains A and B are joined in reverse complementarity, and their second complementary sequences are joined in reverse complementarity. The target sequence specifically targets the 11 bp double-stranded DNA purine-rich strand located 40 bp upstream of the MYC gene P2 promoter. Its specific location within the MYC gene is detailed in [reference needed]. Figure 1 The target sequence can form a DNA triplex with this 11bp sequence.

[0030] The target sequence is shown in SEQ ID NO:1, the first complementary sequence of chain A is shown in SEQ ID NO:2, the second complementary sequence of chain A is shown in SEQ ID NO:3, the first complementary sequence of chain B is shown in SEQ ID NO:4, and the second complementary sequence of chain B is shown in SEQ ID NO:5. The first linker sequence of chains A and B is AGTT, and the second linker sequence of chains A and B is TGAT. Additionally, the target sequence TFO was synthesized for subsequent experiments.

[0031] 2. Cycling reaction

[0032] The lyophilized linear precursor DNA was dissolved in a cyclization buffer solution, as shown in Table 1 of Example 1. The annealing reaction system was as shown in Table 2 of Example 1, and the annealing conditions were: denaturation at 95°C for 5 minutes; 70 cycles of annealing, with the temperature decreasing by 1°C for 1 minute per cycle, cycling until the temperature decreased to 25°C; finally, the temperature was maintained at 4°C to complete the cyclization annealing reaction.

[0033] 3. Purification steps

[0034] Collect the annealing product, add 1 / 10 volume of 3M sodium acetate and an equal volume of pre-cooled isopropanol, and incubate overnight at -20°C. Centrifuge at 12,000 rpm for 10 minutes at 4°C, and discard the supernatant. Wash the precipitate with 70% ethanol, resuspend, centrifuge again, and discard the supernatant. Dissolve the purified DNA precipitate in 30 μL of enzyme-free sterile water and determine the DNA concentration.

[0035] Table 1: Cycloning buffer system

[0036]

[0037]

[0038] Table 2: Annealing reaction system

[0039] reagents system Oligonucleotide chain A (100uM) 20uL Oligonucleotide chain B (100uM) 20uL 5× Annealing Buffer 40uL Enzyme-free sterile water 120uL

[0040] 4. Product Validation

[0041] The cyclic TFO product was verified by electrophoresis (DNA bands were observed and photographed using a UV gel imaging system, and the gel electrophoresis image is shown below). Figure 2 As shown in the figure). Specifically, a 15% non-denaturing polyacrylamide gel was prepared, and DNA molecular weight standard Marker A (25-500 bp), high concentration (300 ng), and low concentration (100 ng) oligonucleotide chains A and B were added to the gel wells respectively (A and B chains had the same concentration, and the total concentration was used as the basis). Figure 3 Cir-TFO (labeled as A+B chain) undergoes cyclization reaction. Figure 3 Cir-TFO marked as unpurified Cir-TFO, Cir-TFO after purification steps ( Figure 3 (Labeled as purified Cir-FTO). Electrophoresis was performed at 120V on ice for 90 minutes. The gel was stained with TBE electrophoresis buffer containing nucleic acid dyes for 30 minutes, and DNA bands were observed and photographed using a UV gel imaging system. Figure 2 The gel electrophoresis image shown illustrates the different migration rates of linear precursor DNA (A+B strand) and circular TFO (Cir-TFO) on the gel, verifying the success of the cyclization reaction.

[0042] Example 2 Cell Experiment

[0043] 1. Cell proliferation experiment

[0044] Human colorectal adenocarcinoma cells (COLO320 DM) in logarithmic growth phase were washed twice with PBS, then detached from the cells using PBS and collected in centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min using a benchtop centrifuge. The supernatant was discarded, and the cells were resuspended in RPMI-1640 complete medium (RMPI-1640 medium + 10% fetal bovine serum + 1% penicillin and streptomycin). The cell suspension was prepared at a ratio of 1.6 × 10⁶ cells / well. 4 One cell line was seeded into two 96-well plates and incubated in a 5% CO2 incubator for 24 hours until the cells adhered. Each well contained 5 μL of DMEM medium, 100 ng of Cir-TFO (prepared according to the method provided in Example 1) or a target sequence TFO (such as secNO1), and 0.16 μL of Lipo8000. TM Transfection reagents were used to prepare transfection mixtures. After replacing the culture medium with fresh medium, DNA-free transfection mixture (control group con), Cir-TFO transfection mixture (Cir-TFO), and target sequence TFO transfection mixture (TFO) were added to each group of cells, respectively. Cells in two 96-well plates were replaced with fresh medium at 48 h and 72 h after drug administration, and 10 μL of CCK-8 reagent was added to each well. After incubation in a 5% CO2 incubator for 1.5 h, the absorbance at 450 nm was measured using a microplate reader. Specific results are shown below. Figure 3 As shown in the figure. Experimental results showed that 48 hours after drug administration, the tumor cell survival rate in the control group (con) was 100%, while the tumor cell survival rates in the TFO group (TFO) and the Cir-TFO group (Cir-TFO) were 100% and 65.8%, respectively. 72 hours after drug administration, the tumor cell survival rate in the control group was 100%, while the tumor cell survival rates in the TFO and Cir-TFO groups were 92.8% and 61.9%, respectively.

[0045] 2. Cell migration experiment

[0046] Colo320 DM cells were spaced at 1.7 × 10⁶ cells per well. 5 Each cell was seeded into a 24-well plate and cultured in a 5% CO2 incubator until 70% confluence was achieved. Add 25 μL of DMEM medium, 500 ng Cir-TFO or target sequence TFO, and 0.8 μL Lipo8000 per well. TMPreparation of transfection reagents: A straight line was drawn in a 24-well plate using a 200 μL pipette tip. After washing twice with PBS, culture medium was added, and the scratched area was photographed using an inverted microscope. After photographing, DNA-free transfection mixture (con), Cir-TFO transfection mixture (Cir-TFO), and target sequence TFO transfection mixture (TFO) were added to each well, respectively. The 24-well plate was incubated in a 5% CO2 incubator, and the scratched area was photographed using an inverted microscope at 24 h and 48 h after drug addition. Cell scratch experiment images are shown below. Figure 4 As shown in Figure -A, cell migration at different time points (0h, 24h, 48h) and in different treatment groups (Con, TFO, Cir-TFO) is observed. In the Con group (control group), the scratch is clear at 0h, with cells distributed on both sides of the scratch. At 24h, cells on both sides of the scratch begin to migrate towards the center, narrowing the scratch width. At 48h, cell migration continues, and the scratch narrows significantly, indicating that the control group cells have a certain migration ability. Compared with the control group, the TFO group shows relatively weaker cell migration towards the center of the scratch at 24h and 48h, and the reduction in scratch width is less significant, indicating that TFO treatment has a certain inhibitory effect on cell migration. In the Cir-TFO group, fewer cells migrate to the center of the scratch at 24h and 48h, and the scratch width reduction is the lowest, indicating that Cir-TFO treatment has a stronger inhibitory effect on cell migration than TFO treatment.

[0047] 3. Cloning experiment

[0048] Human small cell lung cancer cells (H466) in the logarithmic growth phase were washed twice with PBS and then digested with trypsin. The digested cells were collected in centrifuge tubes and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in RPMI-1640 complete medium (RMPI-1640 medium + 10% fetal bovine serum + 1% penicillin and streptomycin). 1000 cells were seeded per well in 6-well plates and incubated in a 5% CO2 incubator for four days. Each well contained 125 μL of LMEM medium, 2.5 μg of circular single-stranded DNA (half A, half B strands) or a target sequence TFO (e.g., secNO1), and 4 μL of Lipo8000. TMPrepare the transfection mixture using the transfection reagents. Replace with fresh medium and add DNA-free transfection mixture (con), Cir-TFO transfection mixture (Cir-TFO), or target sequence TFO transfection mixture (TFO) to each well, respectively. Incubate in a 5% CO2 incubator for 12 days, replacing the medium with fresh medium containing the transfection mixture every 3 days. Discard the medium, wash twice with PBS, and add 1 ml of 4% cell tissue fixative to each well for 20 min. Discard the fixative and wash with PBS, stain with crystal violet for 2 min, wash and air dry, and photograph under white light to obtain the colony formation experiment results, as shown in the figure. Figure 4 As shown in Figure -B, the cell cloning status of the three different treatment groups is illustrated. The number of clones in the TFO group was significantly reduced compared to the Con group, indicating that TFO treatment inhibits cell colony formation. The number of clones in the Cir-TFO group was further reduced, indicating that Cir-TFO treatment has a stronger inhibitory effect on cell colony formation than TFO treatment.

[0049] 3. Example 3: Animal Experiment

[0050] 1. Establishment of animal models of tumors

[0051] Female BALB / c Nude mice aged 6-7 weeks were selected, and colorectal cancer Colo320 DM cells were injected at a rate of 1×10⁻⁶. 6 Inoculate the tumor subcutaneously on the right back of nude mice at a density of 1 mouse / mouse. After inoculation, measure the tumor size of the mice every two days using calipers. When the tumor volume reaches approximately 100 mm², [the tumor is considered complete]. 3 Nude mice were randomly divided into a drug treatment group and a control group, with 6 mice in each group.

[0052] 2. Administration

[0053] Nude mice in the treatment group were administered Cir-TFO (Cir-TFO group) or target sequence TFO (TFO group) via intratumoral injection at a dose of 100 μg / mouse, once every 3 days for a total of 15 days. Nude mice in the control group (PBS group) were injected with an equal volume of PBS buffer.

[0054] 3. Tumor volume measurement

[0055] The major diameter (a) and minor diameter (b) of the tumor were measured with calipers every 3 days, and the result was calculated using the formula V = 1 / 2 × a × b. 2 Tumor volume was calculated. Results showed that after 2 weeks of treatment, the tumor volume in the Cir-TFO group decreased from the initial 100 mm. 3 The length increased to 700mm. 3 Around 1200 mm, the tumor volume in the TFO group increased to 1200 mm. 3 In the control group, the tumor volume increased to 1500 mm. 3The results of the tumor volume growth curve comparison are as follows: Figure 5 As shown. Figure 5 In the linear graph data of tumor volume changes over time, the tumor volume in the PBS group (dotted line) as the control group increased rapidly over time, while the tumor volume in the TFO group (square line) and Cir-TFO group (triangular line) as the treatment groups increased relatively slowly. At 15 days after treatment, the TFO group showed a significant difference compared to the PBS group (*, P<0.05), and the Cir-TFO group showed a highly significant difference compared to the PBS group (****, P<0.0001), indicating that both TFO and Cir-TFO can inhibit tumor growth, with Cir-TFO showing a superior inhibitory effect.

[0056] 4. Toxic and side effect assessment

[0057] After euthanizing the nude mice at the end of the experiment, histopathological examinations were performed on major organs such as the liver, kidneys, lungs, spleen, and heart. The results showed no significant damage to the organs and tissues in the treated group, indicating that this Cir-TFO exhibits high in vitro safety when used to prepare drugs for treating malignant tumors. The results are as follows... Figure 6 As shown.

[0058] 5. Discussion of Results

[0059] Regularly measured tumor volume revealed that tumor growth was significantly inhibited in the treatment group compared to the control group. After 15 days of treatment, the tumor volume in the Cir-TFO group was approximately 50% smaller than that in the control group. Furthermore, histopathological examination of the animals showed that Cir-TFO had no significant toxic side effects on normal tissues in mice, and no significant tissue damage was found within the normal range.

Claims

1. A cyclic triple-stranded oligonucleotide, characterized in that... It consists of two oligonucleotide chains, A and B. Each oligonucleotide chain includes, from the 5' end to the 3' end, a first complementary sequence, a first linker sequence, a target sequence, a second linker sequence, and a second complementary sequence. The first complementary sequences of the two oligonucleotide chains A and B are anticomplementary and linked together, forming a closed circular structure. The target sequence is a TFO sequence that can form a triple-stranded structure with double-stranded DNA in the promoter region of a target gene through Hoogsteen hydrogen bonds. The target gene is selected from proto-oncogenes, anti-apoptotic genes, or tumor metabolism-related genes. The lengths of the first and second complementary sequences are 6-20 bases, and the lengths of the first and second linker sequences are 4-8 bases.

2. The cyclic triple-stranded oligonucleotide as described in claim 1, characterized in that, The target gene is c-MYC; the target sequence is shown in SEQ ID NO:1; the TFO targets the purine-rich or pyrimidine-rich chain in the promoter region of the target gene.

3. The cyclic triple-stranded oligonucleotide as described in claim 1, characterized in that... The first and second complementary sequences of chain A are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively. The first linker sequence of chains A and B is AGTT, and the second linker sequence of chains A and B is TGAT.

4. The method for preparing a cyclic triple-stranded oligonucleotide as described in any one of claims 1-3, characterized in that... Includes the following steps: a) Synthesize oligonucleotide chain A and oligonucleotide chain B respectively; b) Formation of closed ring structures in cyclization buffer via annealing or enzymatic ligation; c) Purify the product to obtain the final product.

5. The method as described in claim 4, characterized in that: The cyclization buffer contains 10-100 mM Tris-HCl (pH 7.0-8.5), 1-10 mM EDTA, 20-200 mM NaCl, and 5-20% glycerol; the annealing procedure is as follows: denaturation at 80-95°C for 1-10 minutes, followed by cooling to 20-40°C at a rate of 0.5-2°C / cycle, for a total of 10-100 cycles.

6. A pharmaceutical composition, characterized in that... It comprises the cyclic triple-stranded oligonucleotide of any one of claims 1-3 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that... The carrier is selected from liposomes, exosomes, polymer nanoparticles or viral vectors, and the pharmaceutical composition is used for injection, oral or topical delivery.

8. The use of the cyclic triple-stranded oligonucleotide according to any one of claims 1-3 in the preparation of tumor-targeting drugs, characterized in that, By targeting and binding to the promoter region of the gene, it induces DNA double-strand breaks, inhibits transcriptional activation, or silences epigenetic information, thereby suppressing tumor cell proliferation, migration, or inducing apoptosis.

9. The application as described in claim 8, characterized in that, The tumor is a malignant tumor.

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