High-stability nucleic acid aptamer and application thereof

Abstract

The invention relates to a high-stability nucleic acid aptamer and application thereof. Specifically, the invention provides a separated nucleic acid molecule, and the nucleic acid molecule is selected from: (1) a nucleic acid molecule as shown in any one or more of SEQ ID NO: 1-4; (2) a nucleic acid molecule hybridized with the sequence limited by (1) under strict conditions; and (3) a nucleic acid molecule having at least 70% or more sequence identity with the sequence defined in (1) or the complementary sequence thereof.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and can be used for the treatment of cancer viruses, etc., specifically involving highly stable nucleic acid aptamers and their applications. Background Technology

[0002] Oligonucleotide sequences, due to their high plasticity and unique three-dimensional structure, can bind to specific targets and are commonly referred to as nucleic acid aptamers. Compared to antibodies, nucleic acid aptamers have many advantages, including low synthesis cost, ease of modification, small molecular weight, high stability, and negligible immune response.

[0003] Nucleolin is a multifunctional protein that is mainly distributed in the nucleolus of cells, and also partially present in the nucleoplasm, cytoplasm, and cell membrane. Studies have demonstrated that nucleolin (NCL) is closely related to multiple physiological processes, including ribosome biosynthesis, chromatin structure, DNA and RNA metabolism, cell division, cell proliferation, angiogenesis, and regulation of apoptosis. Research by F. Tonello et al. has shown that infections caused by viruses such as RSV, HIV, HPIV-3, IAV, EVA71, and CVB are mediated by nucleolin. During angiogenesis in cancerous tissues, nucleolin on the surface of endothelial cell membranes participates in the signal transduction of angiogenesis. Therefore, targeting nucleolin on the surface of endothelial cell membranes will significantly reduce angiogenesis in cancerous tissues, thus becoming a key target for cancer treatment.

[0004] G-quadruplexes are unique structures composed of guanine-rich nucleic acid sequences formed from DNA or RNA sequences. It has been demonstrated that G-quadruplexes are the structural basis for the highly specific binding of nucleic acid sequences to nucleolin. This stable G-quadruplex significantly enhances the binding affinity of aptamers (Apt) to nucleolin.

[0005] Despite their numerous advantages, oligonucleotide aptamers have encountered some obstacles in their progress toward clinical application. Currently, only two aptamers have been approved by the FDA for the treatment of age-related macular degeneration. There are two main reasons for this. First, due to their oligonucleotide nature, these aptamers are relatively unstable, especially in serum, leading to rapid clearance from the bloodstream and poor pharmacokinetic performance. Summary of the Invention

[0006] This invention first provides an isolated nucleic acid molecule, wherein the nucleic acid molecule is selected from:

[0007] (1) Nucleic acid molecules as shown in any one or more of SEQ ID NO: 1-4:

[0008] (2) Nucleic acid molecules that hybridize with the sequence defined in (1) under strict conditions;

[0009] (3) Nucleic acid molecules that have at least 70% sequence identity with the sequence defined in (1) or its complementary sequence.

[0010] A second aspect of the present invention provides an RNA G-quadruplex containing one or more sequences shown in SEQ ID NO: 1-4.

[0011] In one or more embodiments, the G-quadruplex is formed by annealing a nucleic acid molecule containing the sequence.

[0012] In one or more embodiments, a method for preparing the G-quadruplex includes annealing a nucleic acid molecule containing the sequence in a solution containing cations.

[0013] In one or more embodiments, the cation is a divalent cation, such as a potassium ion.

[0014] In one or more embodiments, the molar ratio of the nucleic acid molecule to the cation-containing solution is less than 1:10.

[0015] In one or more embodiments, the G-quadruplex is prepared by mixing a nucleic acid molecule containing the sequence with a solution containing potassium ions, and heating and cooling to form an RNA G-quadruplex.

[0016] In one or more embodiments, the heating and cooling steps are as follows: heating at 90-95°C for 3-7 minutes, and then naturally cooling to room temperature.

[0017] In one or more embodiments, the potassium-containing solution comprises a KCl and K3PO4 buffer pair or a Tris buffer salt.

[0018] In one or more embodiments, the concentration of KCl in the KCl and K3PO4 buffer pair is 80–120 mM, and the concentration of K3PO4 is 5–12 mM.

[0019] In one or more embodiments, the Tris buffer salt concentration is 5–12 mM.

[0020] A third aspect of the present invention provides a pharmaceutical composition comprising (1) a sequence represented by any one or more of SEQ ID NO: 1-4, or an RNA G-quadruplex as described in the second aspect herein, and (2) a pharmaceutically acceptable excipient.

[0021] The fourth invention provides a nucleic acid aptamer containing one or more sequences shown in SEQ ID NO: 1-4, or the nucleic acid aptamer includes an RNA G-quadruplex as described herein.

[0022] In some implementations, the nucleic acid aptamer is able to remain stably in blood-derived samples, preferably for at least 96 hours.

[0023] In some implementations, the blood sample is selected from whole blood, serum, and / or plasma.

[0024] The fifth aspect of the present invention provides the use of RNA G-quadruplexes and / or nucleic acid aptamers as described in any embodiment herein in the preparation of medicaments for treating and / or preventing diseases, said diseases including: (1) diseases that benefit from inhibition of angiogenesis, (2) diseases that benefit from inhibition of vascular endothelial cell circumduction, (3) diseases that benefit from inhibition of vascular endothelial cell migration, (4) diseases that target nucleolin, (5) viral infections, and / or (6) cancer.

[0025] In one or more embodiments, the nucleolin is a cell membrane nucleolin, such as endothelial cell membrane nucleolin or cancer cell membrane nucleolin.

[0026] In some embodiments, the virus includes viruses known in the art that can interact with nucleolin on the cell membrane surface, such as one or more of RSV, HIV, HPIV-3, IAV, EVA71, and CVB.

[0027] In one or more embodiments, the cancer is selected from lung cancer (such as non-small cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma, glioma, head and neck cancer, skin cancer, endometrial cancer, kidney cancer, stomach cancer, small intestine cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, etc. The compositions of the present invention can be used for all stages and types of cancer, including for, for example, minimal residual disease, early-stage cancer, advanced cancer and / or metastatic cancer and / or difficult-to-treat cancer.

[0028] The sixth aspect of the present invention provides a method for improving the binding ability of nucleic acids to nucleolin, the method comprising: contacting the nucleic acid aptamer described herein with nucleolin.

[0029] The seventh aspect of the present invention provides a method for treating and / or preventing diseases, said diseases including: (1) diseases that benefit from inhibiting angiogenesis, (2) diseases that benefit from inhibiting vascular endothelial cell circumduction, (3) diseases that benefit from inhibiting vascular endothelial cell migration, (4) diseases that target nucleolin, (5) viral infections, and / or (6) cancer.

[0030] The eighth aspect of the present invention provides the use of nucleic acid molecules, RNA G-quadruplexes or nucleic acid aptamers as described in any embodiment herein in the preparation of kits for identifying cancer cells or cancer tissues, or for diagnosing cancer.

[0031] In one or more embodiments, the kit contains the nucleic acid molecule or the RNA G-quadruplex.

[0032] In one or more embodiments, the kit further contains a marker.

[0033] In one or more embodiments, the marker is a fluorescent group.

[0034] In one or more embodiments, the fluorescent group is selected from one or more of FAM, HEX, ROX, and Cy5.

[0035] In one or more embodiments, the kit also includes instructions for use.

[0036] In one or more embodiments, the instruction manual describes a method for identifying cancer cells or cancerous tissue, or for diagnosing cancer, using the nucleic acid molecule or RNA G-quadruplex.

[0037] In one or more embodiments, the identification of cancer cells or cancerous tissue includes: contacting the RNA G-quadruplex or nucleic acid aptamer with the cells or tissue to be tested, and identifying cancer cells or cancerous tissue by detecting the RNA G-quadruplex. Preferably, the detection is the detection of a marker conjugated to the RNA G-quadruplex; more preferably, the RNA G-quadruplex is bound to the cell membrane of the cells to be tested.

[0038] The present invention also provides a kit for identifying cancer cells or cancerous tissue, or for diagnosing cancer, the kit comprising the nucleic acid molecule, RNA G-quadruplex, or nucleic acid aptamer described in any embodiment herein.

[0039] In one or more embodiments, the kit further comprises a marker; preferably, the marker is a fluorescent group, such as one or more selected from FAM, HEX, ROX, and Cy5.

[0040] In one or more embodiments, the RNA G-quadruplex is coupled with a marker; preferably, the marker is a fluorescent group, such as one or more selected from FAM, HEX, ROX, and Cy5.

[0041] In one or more embodiments, the kit further includes instructions for use; preferably, the instructions for use describe methods for identifying cancer cells or cancerous tissue, or diagnosing cancer, using the nucleic acid molecule, RNA G-quadruplex, or nucleic acid aptamer.

[0042] In one or more embodiments, the identification is a location detection.

[0043] A ninth aspect of the present invention provides a detection method, the method comprising: contacting the RNAG-quadruplex or nucleic acid aptamer described in any embodiment herein with a cell or tissue to be tested, and identifying cancer cells or cancer tissue by detecting the RNAG-quadruplex.

[0044] In one or more embodiments, the detection is the detection of a marker coupled to the RNA G-quadruplex; preferably, the marker is a fluorescent group.

[0045] In one or more embodiments, the 5' or 3' end of the RNA G-quadruplex is linked to a fluorescent group.

[0046] In one or more embodiments, the fluorescent group is selected from one or more of FAM, HEX, ROX, and Cy5.

[0047] In one or more embodiments, the contact is an incubation at 30–40°C for 0.5–2 hours, more preferably an incubation at 35–37°C for 1–1.5 hours.

[0048] In one or more embodiments, the RNA G-quadruplex binds to the cell membrane of the cell being tested. In one or more embodiments, the cancer cells originate from tissues or organs suffering from cancer or tumors.

[0049] In one or more embodiments, the method includes: linking the RNA G-quadruplex described in any of the embodiments herein to a fluorescent group, contacting it with a cell or tissue to be tested, detecting the binding of the RNA G-quadruplex to the cell or tissue, and identifying cells that detect fluorescence as cancer cells and tissues that detect fluorescence as cancerous tissue.

[0050] In one or more embodiments, the detection method can be used to identify cancer cells or cancerous tissue or to diagnose cancer. Preferably, the identification is a localization detection.

[0051] In some implementations, RNA G-quadruplexes linked to fluorescent groups are injected into the tissue to be tested, and the fluorescently aggregated portion can indicate cancerous tissue containing cancer cells.

[0052] In one or more embodiments, the cancer cells or cancerous tissue originate from cancer selected from lung cancer (such as non-small cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma, glioma, head and neck cancer, skin cancer, endometrial cancer, kidney cancer, stomach cancer, small intestine cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, etc. The compositions of the present invention can be used for all stages and types of cancer, including for, for example, minimal residual disease, early-stage cancer, advanced cancer and / or metastatic cancer and / or difficult-to-treat cancer. Attached Figure Description

[0053] Figure 1 To validate the experimental results of Apt 1 combined with NCL for SPR.

[0054] Figure 2 To validate the experimental results of Apt 2 combined with NCL for SPR.

[0055] Figure 3 To verify the results of the Apt 3 binding NCL assay using IHC-based cell flow cytometry.

[0056] Figure 4 To verify the experimental results of Apt 4 combined with NCL for MST.

[0057] Figure 5 These are the results of a nuclease stability experiment.

[0058] Figure 6 The results are from a cell scratch assay.

[0059] Figure 7 These are the results of a transwell cell invasion experiment.

[0060] Figure 8 The results are from the vascular endothelial cell ringing experiment.

[0061] Figure 9 The results are from an experiment on inhibition of blood vessels in the allantoic membrane of chicken embryos.

[0062] Figure 10 To verify the effectiveness against RSV virus infection.

[0063] Figure 11 To verify the identification of cancer cells.

[0064] Figure 12 Verification for cancer tissue identification. Detailed Implementation

[0065] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0066] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0067] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0068] In this article, "aptamer" refers to oligonucleotide sequences (including DNA and RNA), nucleic acid analogs, or short polypeptides obtained through in vitro screening, which can bind to corresponding ligands or targets with high affinity and strong specificity. For example, nucleic acid aptamers can bind to different targets (including small molecules, ions, small molecules, polypeptides, and even whole cells) with high affinity and strong specificity.

[0069] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0070] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0071] Nucleolin, a multifunctional protein that regulates the expression of various proteins, is predominantly found in the nucleolus in normal cells. However, in cancerous tissues, nucleolin from endothelial cells is extensively relocated to the cell membrane surface and plays a crucial role in mediating angiogenesis-related physiological signals. The inventors have developed a highly stable nucleic acid aptamer with low cytotoxicity in plasma, targeting cell membrane surface nucleolin (NCL). This aptamer binds to nucleolin on the cell membrane surface but does not enter the cell, thereby blocking cell membrane nucleolin-mediated angiogenesis. Given the need for abundant new blood vessels in cancerous tissues, this aptamer can be used for cancer treatment. Furthermore, because the aptamer does not enter the cell and does not interfere with the function of nucleolin within the cell, it exhibits low cytotoxicity. Considering the important role of nucleolin in viral infections, such as RSV, important viruses interact with nucleolin on the cell surface to regulate attachment and invasion processes. The aptamer of this invention can also be applied to treat viral infections such as RSV, HIV, HPIV-3, IAV, EVA71, and CVB. This completes the present invention.

[0072] This invention provides an isolated nucleic acid molecule selected from: (1) nucleic acid molecules shown in any one or more of SEQ ID NO: 1-4; (2) nucleic acid molecules that hybridize with the sequence defined in (1) under stringent conditions; and (3) nucleic acid molecules that have at least 70% sequence identity with the sequence defined in (1) or its complementary sequence. In an exemplary embodiment, the nucleotide sequences of the nucleic acid molecule are as follows: the sequence of the nucleic acid molecule forming Apt 1 is shown as 5′-GUUAGGGU-3′ (SEQ ID NO: 1), the sequence of the nucleic acid molecule forming Apt 2 is shown as 5′-GUUAGGGUU-3′ (SEQ ID NO: 2), the sequence of the nucleic acid molecule forming Apt 3 is shown as 5′-GUUAGGGUUA-3′ (SEQ ID NO: 3), and the sequence of the nucleic acid molecule forming Apt 4 is shown as 5′-GUUAGGGUUA-3′ (SEQ ID NO: 4).

[0073] In this invention, “strict conditions” refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the similarity between the two sequences is at least 90%, preferably more than 95%.

[0074] RNA G-quadruplexes can be prepared from the RNA molecules of the present invention. Therefore, the present invention also provides an RNA G-quadruplex containing any one or more of the sequences shown in SEQ ID NO: 1-4.

[0075] The RNA G-quadruplex of the present invention can be a nucleic acid aptamer (Apt). Therefore, in some embodiments, the present invention also provides a nucleic acid aptamer containing the sequence shown in any one or more of SEQ ID NO: 1-4.

[0076] The preparation method of G-quadruplexes can be a commonly used thermal annealing process in the art, assembling a single-stranded nucleic acid sample with a designed sequence into a G-quadruplex. For example, the nucleic acid molecule containing the designed sequence is annealed in a solution containing cations. The cation can be a divalent cation, such as potassium ions. The molar ratio of the nucleic acid molecule to the solution containing the cation is less than 1:10. In an exemplary embodiment, the single-stranded nucleic acid sample is mixed with KCl and K3PO4 solutions, heated for annealing, and then naturally cooled to room temperature to form a G-quadruplex. In the annealing procedure, the annealing time and temperature are selected according to the assembly requirements of the RNA three-dimensional structure, for example, heating at 90-95°C for 3-7 minutes, followed by natural cooling to room temperature to complete the assembly of the G-quadruplex. The preparation of the single-stranded nucleic acid sample can be carried out using methods commonly used in the art, such as solid-phase synthesis. It should be understood that when a potassium ion solution is used to mix with the single-stranded nucleic acid sample, the center of the plane of the prepared G-quadruplex is composed of potassium ions.

[0077] In some embodiments, the G-quadruplex or nucleic acid aptamer of the present invention is prepared by the following method: synthesizing the sequence shown in any one of SEQ ID NO: 1-4 using solid-phase synthesis, and combining the synthesized nucleic acid with a K-containing... + The solutions are mixed and heated to anneal to form G-quadruplexes or nucleic acid aptamers. Specifically, nucleic acid molecules with the sequence shown in SEQ ID NO:1 are annealed to form G-quadruplex Apt 1, nucleic acid molecules with the sequence shown in SEQ ID NO:2 are annealed to form G-quadruplex Apt 2, nucleic acid molecules with the sequence shown in SEQ ID NO:3 are annealed to form G-quadruplex Apt 3, and nucleic acid molecules with the sequence shown in SEQ ID NO:4 are annealed to form G-quadruplex Apt 4.

[0078] In some implementations, the K-containing + The solution contains a KCl and K3PO4 buffer pair or a Tris buffer salt. Preferably, the KCl concentration in the KCl and K3PO4 buffer pair is 80–120 mM, the K3PO4 concentration is 5–12 mM, and the Tris buffer salt concentration is 5–12 mM.

[0079] The RNA G-quadruplex of this invention is thermodynamically stable, exhibits high specificity for nucleolin on the cell membrane, and does not internalize into the cell. This RNA G-quadruplex can be used to target nucleolin, a marker of tumor cells. The inventors have also discovered that this G-quadruplex can inhibit vascular endothelial cell migration, inhibit vascular endothelial cell circulation, and / or inhibit angiogenesis.

[0080] Therefore, the present invention also provides a pharmaceutical composition comprising the sequence shown in any one or more of SEQ ID NO: 1-4, or the RNA G-quadruplex described in any embodiment herein, and pharmaceutically acceptable excipients.

[0081] In this invention, a "pharmaceuticalally acceptable excipient" is a pharmaceutically or food-grade carrier, solvent, suspending agent, or excipient used to deliver the formulation or pharmaceutical composition of this invention to an animal or human. As used herein, a pharmaceutically acceptable excipient is non-toxic to the recipient of the composition at the dosage and concentration employed. It may include various types of carriers or excipients commonly used in therapeutics to deliver proteins, as is known in the art. Exemplary excipients may be liquids or solids, including but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, flow aids, sweeteners, dyes / coloring agents, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers. In some embodiments, a pharmaceutically acceptable excipient may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, sprays, compressed air or other suitable gases, or other suitable inactive ingredients used in conjunction with a pharmacodynamic compound. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition, ed. ARGenrmo, 1990, Mack Publishing Company. The optimal pharmaceutical composition can be determined based on the intended route of administration, delivery method, and required dosage.

[0082] The pharmaceutical compositions described herein are suitable for one or more administration or delivery methods selected from the group consisting of: inhalation via respiratory tract nebulization, nasal drops, oral administration, direct injection (e.g., intravenous injection), subcutaneous injection, intradermal injection, intramuscular injection, and mucosal administration. In an exemplary embodiment, the pharmaceutical compositions described herein are administered via tail vein injection.

[0083] This invention also provides the use of nucleic acid molecules, RNA G-quadruplexes, and / or nucleic acid aptamers as described in any embodiment herein in the preparation of medicaments for treating and / or preventing diseases, including one or more selected from: (1) diseases that benefit from inhibiting angiogenesis, (2) diseases that benefit from inhibiting vascular endothelial cell circumduction, (3) diseases that benefit from inhibiting vascular endothelial cell migration, (4) diseases targeting nucleolin, (5) viral infections, and (6) cancer. In some embodiments, the nucleolin is endothelial cell membrane nucleolin.

[0084] In some embodiments, the virus includes one or more of the following that are inhibited in the art and can interact with nucleolin on the cell membrane surface: RSV, HIV, HPIV-3, IAV, EVA71, and CVB.

[0085] In some embodiments, the diseases include various solid tumors, including but not limited to lung cancer (such as non-small cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma, glioma, head and neck cancer, skin cancer, endometrial cancer, kidney cancer, stomach cancer, small intestine cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, etc. The nucleic acid aptamers or pharmaceutical compositions of the present invention can be used for all stages and types of cancer, including for, for example, minimal residual disease, early-stage cancer, advanced cancer and / or metastatic cancer and / or difficult-to-treat cancer.

[0086] This invention also provides the use of nucleic acid molecules, RNA G-quadruplexes, or nucleic acid aptamers as described in any embodiment herein in the preparation of kits for the identification of cancer cells or cancerous tissues, or for the diagnosis of cancer. In this document, the kits may also contain markers, which may be fluorescent groups, such as one or more selected from FAM, HEX, ROX, and Cy5.

[0087] In some embodiments, the kits described herein also include instructions for use. These instructions describe methods for identifying cancer cells or cancerous tissue, or for diagnosing cancer, using the nucleic acid molecules or RNA G-quadruplexes described herein. In some embodiments, identifying cancer cells or cancerous tissue includes: contacting the RNA G-quadruplex or nucleic acid aptamer with test cells or test tissue, and identifying cancer cells or cancerous tissue by detecting the RNA G-quadruplex. In some embodiments, the detection may be the detection of a marker conjugated to the RNA G-quadruplex. In some embodiments, the RNA G-quadruplex binds to the cell membrane of the test cells.

[0088] In some embodiments, the present invention also provides a kit for identifying cancer cells or cancerous tissue, or for diagnosing cancer, said kit comprising a nucleic acid molecule, an RNA G-quadruplex, or a nucleic acid aptamer. In this document, the kit may also contain a marker, which may be a fluorescent group, such as one or more selected from FAM, HEX, ROX, and Cy5. In some embodiments, the RNA G-quadruplex is coupled with a marker.

[0089] In some embodiments, the kit also includes instructions for use. In some embodiments, the instructions for use describe methods for identifying cancer cells or cancerous tissue, or diagnosing cancer, using the nucleic acid molecule, RNA G-quadruplex, or nucleic acid aptamer. In some embodiments, identification is a localization detection.

[0090] This invention also provides a method for identifying cancer cells or cancerous tissue, the method comprising: contacting an RNA G-quadruplex or a nucleic acid aptamer with a cell or tissue to be tested, and identifying cancer cells or cancerous tissue by detecting the RNA G-quadruplex, wherein the RNA G-quadruplex and nucleic acid aptamer are as described herein. In some embodiments, the detection may be the detection of a marker conjugated to the RNA G-quadruplex. In some embodiments, the marker is a fluorescent group. In some embodiments, the RNA G-quadruplex binds to the cell membrane of the cell to be tested. In some embodiments, the identification is a localization detection.

[0091] This invention also provides a localization detection method, the method comprising: linking the RNAG-quadruplex described in any embodiment of this invention to a fluorescent group, contacting it with a cell or tissue to be tested, detecting the binding of the RNAG-quadruplex to the cell or tissue, and identifying cells that detect fluorescence as cancer cells and tissues that detect fluorescence as cancerous tissue. The localization refers to determining the location of cancer cells or cancerous tissue within the body, for example, within a specific organ.

[0092] In one or more embodiments, the 5' or 3' end of the RNA G-quadruplex is linked to a fluorescent group. In this document, the RNA G-quadruplex linked to the fluorescent group is injected into the body, and the fluorescently aggregated portion is cancerous tissue. Incubation can be performed under commonly used cell incubation conditions in the art, such as incubation at 30–40°C for 0.5–2 h, more preferably at 35–37°C for 1–1.5 h. In one or more embodiments, the detection is the detection of the fluorescent group contained in the RNA G-quadruplex. In one or more embodiments, the RNA G-quadruplex binds to the cell membrane of the cell being tested.

[0093] In some implementations, RNA G-quadruplexes linked to fluorescent groups are injected into the tissue to be tested, and the fluorescently aggregated portion can indicate cancerous tissue containing cancer cells.

[0094] In one or more embodiments, the cancer cells originate from cancer selected from lung cancer (such as non-small cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma, glioma, head and neck cancer, skin cancer, endometrial cancer, kidney cancer, stomach cancer, small intestine cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, etc. The compositions of the present invention can be used for all stages and types of cancer, including for, for example, minimal residual disease, early-stage cancer, advanced cancer and / or metastatic cancer and / or difficult-to-treat cancer.

[0095] The present invention also provides the use of one or more of the nucleic acid molecules and / or G-quadruplexes described in any embodiment herein in the preparation of kits for identifying cancer cells, detecting cancer tissue, or diagnosing cancer.

[0096] In some embodiments, the kit includes: a nucleic acid molecule and / or RNA G-quadruplex as described in any embodiment herein, a fluorescent group, and instructions for use. The kit is used to detect cancer cells, tissues or organs with tumors or cancer. The fluorescent group can be one or more commonly used in the art, such as FAM, HEX, ROX, Cy5, etc. The instructions for use describe the detection method as described herein.

[0097] The present invention has the following beneficial effects:

[0098] Compared with existing nucleic acid aptamers, the present invention can remain stable in plasma for more than 96 hours and has good nucleolin binding ability and low cytotoxicity.

[0099] The present invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and reagents used in the embodiments are conventional methods and reagents in the art.

[0100] method

[0101] 1. Solid-phase synthesis method

[0102] Solid-phase synthesis method is referenced in the literature (Troy AW et al., (2002) Bioconjugate Chem. 13: 1155-58).

[0103] 2. SPR, MST, and IHC combined with flow cytometry analysis

[0104] 2.1 The binding ability of SPR to nucleolin was analyzed. The experimental method is as shown in the literature (Chao DX et al., (2021) Front Chem.9:781198; Chao DX et al. (2023) Int J Mol Sci.,24(18):14344).

[0105] The specific steps for combining SPR analysis are as follows: The running buffer for the chip coupling process is HEPES-NaCl. The chip surface is activated by mixing equal volumes of 0.4M EDC and 0.1M NHS, with an activation volume of 170 μL and a flow rate of 20 μL / min. Then, NCL is coupled at pH 4.0, a concentration of 25 ng / ml, a volume of 340 μL, and a flow rate of 20 μL / min. Finally, it is blocked with 1M ethanolamine, with a volume of 170 μL and a flow rate of 20 μL / min. (The process is repeated twice in the original text.) Apt 1 analyte at concentrations of 0 nM, 25 nM, 74 nM, 222 nM, 667 nM, 2 μM, and 6 μM was sequentially injected in 60 μL injection volumes. Regeneration was performed between concentrations using 1 M KCl in 30 μL volumes. The response curves for each concentration were recorded.

[0106] 2.2 MST analysis of the binding affinity of the RNA G-quadruplex to nucleolin was performed as follows: NCL protein at an initial concentration of 4 μM was serially diluted and co-incubated with 0.6 μM aptamer for 2 min. The sample was then loaded into a nanothermal glass capillary tube, and microthermal electrophoresis was performed using 80% LED power and 80% MST. Kd values ​​were measured from repeated readouts using the mass action equation and nanothermal software.

[0107] 2.3 IHC combined with flow cytometry to verify the binding of the RNA G-quadruplex to nucleolin. The steps are as follows: Apt 3-G-quadruplex prepared from Apt 3 competes with nucleolin antibody and cancer cells, such as HeLa cells and MCF7 cells, are stained. Flow cytometry is then used to detect whether Apt 3-G-quadruplex can bind to nucleolin on the surface of cancer cell membranes. The method is as follows: Cells are cultured in 10% DMEM medium, and then washed twice with pre-cooled PBS to remove the culture medium. Cells are collected after digestion with 2 mmol / L EDTA. After incubation at 4°C with AS1411-FAM or G-quadruplex Apt 3 (1-52 μmol / L) for 30 min, the cells are washed with PBS, and then incubated at 4°C with different concentrations of nucleolin antibody (1 μg, 0.5 μg, 0.1 μg) for 1 h. Then, an appropriate amount of fluorescent secondary antibody against nucleolin antibody is added, mixed thoroughly, and incubated at room temperature in the dark for 30 min. After resuspending the cells in 200 μL of pre-cooled PBS, the fluorescence intensity was recorded using a flow cytometer.

[0108] 3. Nuclease stability experiment

[0109] A 10 μL sample of aptamer was incubated with an equal volume of culture medium containing fetal bovine serum at 37°C for 0 to 96 hours. Subsequently, 1.5 volumes of 10M urea were added to the incubation mixture. The entire sample was then denatured at 100°C for 5 minutes and subsequently stored at -20°C. Finally, the sample was analyzed by 20% denaturing polyacrylamide gel electrophoresis using GelStar. TM Staining is used to visualize the resulting bands.

[0110] 4. Verification of anti-angiogenic effect

[0111] 4.1 After culturing human umbilical vein endothelial cells (HUVECs) to 90% confluence in 6-well plates, wound sutures were created using 200 μL plastic pipette tips. Cells were then incubated in serum-free ECM medium and divided into two groups: a VEGF-added group (10 ng / mL) and a VEGF-unadded group. The VEGF-added groups were exposed to low-dose (10 μM) or high-dose (20 μM) Apt 1 samples, a mutant negative control sample (20 μM), and a positive control nucleolin-specific aptamer AS1411 (10 μM), respectively. The control group was treated with an equal volume of serum-free ECM medium. Wound healing was recorded at time points 0, 24, and 48 hours, and cell migration areas were quantified by image analysis.

[0112] 4.2 Cell migration experiments were performed using the Transwell cell migration system (8 μm pore size, BD Falcon, MA, USA). HUVECs (2 × 10⁶ cells / well) were used. 4 Cells were added to the upper chamber of a Transwell plate, while the lower chamber contained 500 μL of serum-free culture medium with or without VEGF (10 ng / mL), as well as Apt 1 sample (low dose 10 μM / high dose 20 μM), mut sample (20 μM), and AS1411 (10 μM). After incubation for 48 hours, the migrated cells were fixed with 95% methanol, stained with 0.1% crystal violet for 30 minutes, and then washed five times with PBS.

[0113] 4.3 50 μL of Matrigel was coated into the wells of each 96-well plate. Subsequently, after the Matrigel solidified, HUVECs (1 × 10⁻⁶ per well) were applied. 4 Cells were dispersed in wells. Cells in the experimental group were treated with or without vascular endothelial growth factor (10 ng / mL) and exposed to low-dose (10 μM) or high-dose (20 μM) Apt 1 sample, a solid-phase synthesized mutant sample (Mutant: GUUAAAAU) (20 μM), and AS1411 (10 μM). The control group was treated with an equal volume of serum-free ECM medium. Cells were incubated at 37°C and 5% CO2 for 6 hours, and then images were taken using an optical microscope.

[0114] 4.4 The anti-angiogenic effect was studied using a chicken embryo chorionic villus (CAM) angiogenesis model. Leghorn fertilized eggs (Greek Pindos) were incubated at 37°C for 9 days. Subsequently, a window was made in the eggshell to expose the CAM, and the CAM was covered with sterile tape before the eggs were returned to the incubator. On day 12 of embryonic development, 100 μL of distilled water containing only distilled water (as a control) or distilled water containing low (10 μM) or high (20 μM) doses of Apt 1, Mutant (20 μM), and AS1411 (10 μM) was applied to a 1 cm² area of ​​the CAM within the silica ring. After incubation at 37°C for 48 hours, photographs were taken using a microscope equipped with a digital camera, and the total area of ​​blood vessels was measured using Image Proplus image analysis software. Each sample was tested three times, with 5–10 eggs used for each data point.

[0115] 5. Verification of anti-RSV virus infection effect

[0116] The anti-RSV activity of the RNA G-quadruplex was evaluated using an MTT assay to simulate infected cells.

[0117] 6. Cancer tissue identification and verification

[0118] The cancer tissue recognition ability of the RNA G-quadruplex linked to a fluorescent group was evaluated by in vivo imaging through small animal tumor tissue transplantation.

[0119] The steps are as follows: After 3-4 weeks of acclimatization feeding of null / null mice, feed them 1×10⁻⁶ mice per week. 7 One HeLa cell was subcutaneously seeded into the back of a nude mouse. When the tumor volume reached 1.0 cm, 100 μL of the 3'-terminal Cy5-labeled aptamer of this study was injected via the tail vein to a final concentration of 22.5 μM. Fluorescence images were collected 30 min later using an in vivo imaging system.

[0120] 7. Cell staining

[0121] Cells: Cancer cells (MCF7) and normal cells (LX2).

[0122] Steps: Cells (1×10⁻⁶) 5 Cells were seeded into 6-well plates pre-inserted with coverslips and incubated at 37°C in a 5% CO2 incubator for 12-16 hours until cell adhesion was achieved. The culture medium was then discarded, and 600 μL of 1 μM Apt 4-G-tetramolecularly linked Cy5 was added and incubated at 37°C for 1 hour. The liquid in the wells was discarded, and 1 mL of PBS was added to wash away any remaining liquid. The wells were then incubated at 37°C for 3 minutes with 1 mL of PBS to remove non-specific binding. The wells were then washed again with 1 mL of PBS. The cells were fixed with 1 mL of 4% paraformaldehyde at room temperature for 20 minutes. After washing three times with PBS, 1 mL of DAPI (10 μg / mL, Bioworld) staining solution was added and stained at room temperature for 10 minutes. After washing three times with PBS, 1 mL of DID (10 μg / mL, Beyotime) staining solution was added and stained at 37°C for 20 minutes. After washing three times with PBS, the slides were mounted with anti-fluorescence quenching mounting medium and then placed under a laser confocal microscope to record images.

[0123] Example 1: Preparation of G-quadruplex and analysis of its binding ability with nucleolin

[0124] The sequence of SEQ ID NO:1 was synthesized using a solid-phase synthesis method. After sequence synthesis, 10 μM of the nucleic acid sample was mixed with a solution containing 100 mM KCl and 10 mM K3PO4 (pH 7.0), heated at 95 °C for 5 minutes, and then allowed to cool naturally to room temperature to form a G-quadruplex (aptamer Apt 1). Similarly, Apt 2 (annealed using SEQ ID NO:2), Apt 3 (annealed using SEQ ID NO:3), and Apt 4 (annealed using SEQ ID NO:4) were prepared using the same method. Mutant samples, when synthesized using the same solid-phase method, failed to form a G-quadruplex structure.

[0125] (1) The results of the SPR experiment show that ( Figure 1 and Figure 2 The aptamers Apt 1 and Apt 2 can bind to nucleolin (NCL), and their Ka values ​​are in the nmol range.

[0126] (2) IHC combined with cell loss counting to verify the binding of RNA G-quadruplex to nucleolin.

[0127] like Figure 3 The Apt 3G-tetrachain compound shown can antagonize nucleolin antibodies and bind nucleolin on the surface of cancer cell membranes. The results for Apt 1, Apt 2, and Apt 4 are similar to those for Apt 3.

[0128] (3) MST verification of the binding of the RNA G-quadruplex to nucleolin

[0129] like Figure 4 The Apt 4G-tetrachain shown can bind to nucleolin (NCL), and the Ka values ​​of both are on the order of nmol.

[0130] Example 2: Nuclease Stability Experiment

[0131] The results of Apt 1 assays showed that Apt 1 exhibited good resistance to nucleases in serum, and its concentration remained above 90% after 96 hours. Figure 5 The results for Apt 2, Apt 3, and Apt 4 are similar to those for Apt 1.

[0132] Example 3: Verification of anti-angiogenic effect

[0133] like Figure 3 , Figures 6 to 8 As shown, Apt 1 and the positive control AS1411 significantly inhibited the migration and circumduction of human umbilical vein endothelial cells (HUVECs), while the negative control (mutant group) did not. The results indicate (as shown in...) Figure 9 As shown in the figure, Apt1 has a significant inhibitory effect on angiogenesis. The results for Apt2, Apt3, and Apt4 are similar to those for Apt1.

[0134] Example 4: Verification of anti-RSV virus infection effect

[0135] This embodiment used a series of concentrations of Apt 1 (0.78 μM to 50 μM) as treatment groups. In the RSV infection group, cell viability was significantly reduced to approximately 70% (M group, mutant group). However, in the treatment groups, with increasing Apt 1 concentration, the cell viability of RSV-infected cells gradually improved in a dose-dependent manner, eventually recovering to the control group level (C group, control blank group) (see...). Figure 10 The results for Apt2, Apt3, and Apt4 are similar to those for Apt1.

[0136] These results indicate that Apt 1 through Apt 4 can bind to NCL on the cell membrane, preventing RSV entry and thus preventing RSV infection.

[0137] Example 5: Verification of Cancer Cell Identification

[0138] This example uses Apt 4-Cy5 staining to differentiate cancer cells from normal cells. Figure 11 As shown, normal cells (LX2) could not be stained after co-culturing with AP 4-Cy5, but the cell membrane of cancer cells (HeLa) could be clearly observed. The results for Apt 1, Apt 2, and Apt 3 were similar to those for Apt 4.

[0139] Example 6: Verification of Cancer Tissue Identification

[0140] This embodiment uses Apt 4-Cy5 injected via the tail vein into a nude mouse HeLa tumor model, such as... Figure 12 As shown, tumor tissue enrichment fluorescence was observed 30 minutes after sample injection, while no tumor tissue enrichment fluorescence was observed when the mutant sequence (GUUATTT UUA) linked to Cy5 was injected into mice. The results for Apt 1, Apt 2, and Apt 3 were similar to those for Apt 4.

[0141] Part of the sequence in this article

[0142] >SEQ ID NO: 1Apt 1

[0143] GUUAGGGU

[0144] >SEQ ID NO: 2Apt 2

[0145] GUUAGGGUU

[0146] >SEQ ID NO: 3Apt 3

[0147] GUUAGGGUUA

[0148] >SEQ ID NO: 4Apt 4

[0149] GUUAGGGUUA

[0150] >SEQ ID NO: 5 mutant sample

[0151] GUUATTTUUA

Claims

1. An isolated nucleic acid molecule, said nucleic acid molecule being selected from: (1) Nucleic acid molecules as shown in any one of SEQ ID NO: 1-4; (2) Nucleic acid molecules that hybridize with the sequence defined in (1) under strict conditions; (3) Nucleic acid molecules that have at least 70% sequence identity with the sequence defined in (1) or its complementary sequence.

2. An RNA G-quadruplex, said RNA G-quadruplex containing the sequence shown in any one of SEQ ID NO: 1-4; Preferably, the G-quadruplex is formed by annealing a nucleic acid molecule containing the sequence; More preferably, the method of making the G-quadruplex comprises: The nucleic acid molecule containing the sequence is annealed in a solution containing cations.

3. The RNA G-quadruplex as described in claim 2, characterized in that, The method for preparing the G-quadruplex has one or more of the following characteristics: The annealing step is as follows: heating at 90-95°C for 3-7 minutes, and then cooling to room temperature; The cation is a divalent cation, such as a potassium ion; The molar ratio of the nucleic acid molecules to the cation-containing solution is less than 1:

10.

4. A pharmaceutical composition comprising (1) the sequence shown in any one of SEQ ID NO: 1-4 or the RNA G-quadruplex as described in claim 2 or 3, and (2) a pharmaceutically acceptable excipient.

5. A nucleic acid aptamer, said nucleic acid aptamer containing the sequence shown in any one of SEQ ID NO: 1-4, or said nucleic acid aptamer comprising the RNA G-quadruplex as described in claim 2 or 3. Preferably, the nucleic acid aptamer is stable in blood-derived samples. Preferably, the blood-derived sample is selected from whole blood, serum, and / or plasma.

6. The use of the nucleic acid molecule of claim 1, the RNA G-quadruplex of claim 2 or 3, or the nucleic acid aptamer of claim 5 in the preparation of a medicament for treating and / or preventing a disease, wherein the disease includes one or more of the following: (1) a disease that benefits from inhibiting angiogenesis, (2) a disease that benefits from inhibiting vascular endothelial cell circumduction, (3) a disease that benefits from inhibiting vascular endothelial cell migration, (4) a disease that targets nucleolin, (5) a viral infection, and (6) cancer; Preferably, the virus includes one or more of RSV, HIV, HPIV-3, IAV, EVA71, and CVB; Preferably, the cancer is selected from lung cancer, colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma, glioma, head and neck cancer, skin cancer, endometrial cancer, kidney cancer, stomach cancer, small intestine cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, and thyroid cancer.

7. Use of the nucleic acid molecule of claim 1, the RNA G-quadruplex of claim 2 or 3, or the nucleic acid aptamer of claim 5 in the preparation of a kit, wherein the kit is used to identify cancer cells or cancer tissue, or to diagnose cancer; Preferably, the kit further contains a marker; more preferably, the marker is a fluorescent group, such as one or more selected from FAM, HEX, ROX, and Cy5. Preferably, the kit further includes an instruction manual; more preferably, the instruction manual describes a method for identifying cancer cells or cancer tissue, or diagnosing cancer, using the nucleic acid molecule or RNA G-quadruplex.

8. The use as described in claim 7, characterized in that, The identification of cancer cells or cancerous tissue includes: contacting the RNA G-quadruplex or nucleic acid aptamer with the cells or tissue to be tested, and identifying cancer cells or cancerous tissue by detecting the RNA G-quadruplex. Preferably, the detection is the detection of a marker coupled to the RNA G-quadruplex. Preferably, the RNA G-quadruplex binds to the cell membrane of the cell to be tested.

9. A kit for identifying cancer cells or cancerous tissue, or for diagnosing cancer, said kit comprising the nucleic acid molecule of claim 1, the RNA G-quadruplex of claim 2 or 3, or the nucleic acid aptamer of claim 5. Preferably, the kit further contains a marker; more preferably, the marker is a fluorescent group, such as one or more selected from FAM, HEX, ROX, and Cy5. Preferably, the RNA G-quadruplex is coupled with a marker; more preferably, the marker is a fluorescent group, such as one or more selected from FAM, HEX, ROX, and Cy5. Preferably, the kit further includes an instruction manual; more preferably, the instruction manual describes a method for identifying cancer cells or cancerous tissue, or diagnosing cancer, using the nucleic acid molecule, RNA G-quadruplex, or nucleic acid aptamer. Preferably, the identification is a location detection.

10. A method for identifying cancer cells or cancerous tissue, characterized in that, The method includes: contacting an RNA G-quadruplex or a nucleic acid aptamer with a cell or tissue to be tested, and identifying cancer cells or cancer tissue by detecting the RNA G-quadruplex, wherein the RNA G-quadruplex is as described in claim 2 or 3, and the nucleic acid aptamer is as described in claim 5. Preferably, the detection is the detection of a marker coupled to the RNA G-quadruplex; more preferably, the marker is a fluorescent group. Preferably, the RNA G-quadruplex binds to the cell membrane of the cell being tested. Preferably, the identification is a location detection.

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