Nucleic acid aptamer aiming at ricin and / or abrus precatorius toxin and application of nucleic acid aptamer

Abstract

The invention relates to the technical field of biological medicine, in particular to a nucleic acid aptamer for ricin and / or abrus precatorius toxin and application of the nucleic acid aptamer. Based on a toxicity damage mechanism of ricin and abrus precatorius toxin, starting from reasonable drug design, a nucleic acid chemical modification strategy is adopted, a nucleic acid aptamer with a stable stem-loop conformation is designed, and the adopted sequence design and modification core strategy comprises but is not limited to locked nucleic acid modification of a conserved region GAGA; a stable conformation design (a plurality of CG units) of a'stem 'region on both sides of the'loop'; performing sulfo-modification on a sequence skeleton; the tail end of the sequence is modified by chemical functional groups (such as cholesterol and biotin). The nucleic acid aptamer provided by the invention has good in-vivo and in-vitro pharmacodynamic activity, and can realize remarkable animal protection rate and prolonged survival time under lethal dose.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nucleic acid aptamer for ricin and / or absinthecetine and its application. Background Technology

[0002] Ricin is a natural plant toxin extracted from castor beans and belongs to the type II ribosome-inactivating protein (RIP). Its molecular structure consists of two chains, A and B, linked by disulfide bonds: the B chain binds to receptors containing galactose residues (such as glycoproteins and glycolipids) on the cell surface, mediating endocytosis; the A chain possesses highly efficient glycosidase activity, irreversibly catalyzing the depurination of specific adenine sites on eukaryotic ribosome 28S rRNA after entering the cytoplasm, thereby inducing protein synthesis failure and ultimately activating cell death pathways.

[0003] Ricin is extremely toxic; its median lethal dose (LD50) for humans is... 50 The lethal dose (RTA) is approximately 0.1-1.0 µg / kg body weight; even a trace amount can be fatal. Currently, there is no specific antidote for ricin poisoning in clinical practice; treatment is limited to symptomatic relief. Antidote strategies in research mainly include: 1) Small molecule inhibitors: designed targeting the active site of the RTA, but these generally suffer from weak affinity and poor cell penetration; 2) Neutralizing antibodies: currently the most effective countermeasure, with several monoclonal antibodies showing good protective effects in animal models. However, antibodies have drawbacks such as high production costs, poor stability, and potential immunogenicity.

[0004] Abrin is a highly toxic protein extracted from the seeds of Abrus precatorius, a legume vine. It is currently the most potent known plant toxin. Its molecular structure consists of two chains, A and B, linked by disulfide bonds. The B chain is responsible for binding to receptors containing galactose residues (such as glycoproteins and glycolipids) on the cell surface, mediating the endocytosis of the toxin. The A chain has highly efficient glycosidase activity, which, after entering the cytoplasm, irreversibly catalyzes the depurination of specific adenine sites on the 28S rRNA of eukaryotic ribosomes, thereby inducing protein synthesis failure and ultimately activating cell death pathways.

[0005] Abrus precatorius toxin is extremely toxic, with a median lethal dose (LD50) in mice. 50The lethal dose for humans is 0.1-1.0 μg / kg, with a concentration of 0.04 μg / kg. Currently, there is no specific antidote for russet toxin poisoning in clinical practice; treatment is limited to symptomatic relief. Antidote strategies in research mainly include: 1) Small molecule inhibitors: designed targeting the active site of the ricin-releasing antigen (RTA), but these generally suffer from weak affinity and poor cell penetration; 2) Neutralizing antibodies: currently the most effective countermeasure, with several monoclonal antibodies showing good protective effects in animal models. However, antibodies have drawbacks such as high production costs, poor stability, and potential immunogenicity. Therefore, developing highly efficient and specific antidotes for ricin and russet toxin is of paramount importance for national security, public health emergency response, and clinical treatment.

[0006] Nucleic acid aptamers are single-stranded DNA or RNA molecules screened from nucleic acid sequence libraries using SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology. They are often referred to as "chemical antibodies" due to their high affinity and specificity in binding to targets. Compared to antibodies, they offer many advantages, such as a wider target molecule range, better stability, ease of modification, and low immunogenicity. In recent years, nucleic acid aptamers have shown great potential in the detection and detoxification research of abrinogen toxin due to their unique advantages. Several nucleic acid aptamer molecules have been found to bind efficiently to abrinogen toxin. However, nucleic acid aptamers are mostly used for detection purposes, with relatively few drug studies targeting antitoxin, and no broad-spectrum inhibitors have been reported. Furthermore, issues such as low cellular uptake, poor anti-enzymatic stability, and short circulation time need to be addressed.

[0007] Therefore, in response to the real threats posed by ricin and absinthes, developing safe and effective novel nucleic acid aptamer antidotes is a major need in the field of public safety, and has extremely important social significance and clinical application value. Summary of the Invention

[0008] The purpose of this invention is to provide a nucleic acid aptamer for ricin and / or absinthecetine and its application, in order to solve the problems existing in the prior art.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] The present invention provides a nucleic acid aptamer for ricin and / or absinthecin, wherein the nucleotide sequence of the nucleic acid aptamer is shown as any one or more sequences in SEQ ID NO.1-SEQ ID NO.9.

[0011] This invention provides the use of the above-described nucleic acid aptamers in the preparation of products for detecting ricin and / or absinthecin.

[0012] Optionally, the product includes reagents, reagent kits, and chips.

[0013] The present invention provides a product for detecting ricin and / or abrinogen toxin, the product comprising the above-mentioned nucleic acid aptamers.

[0014] This invention provides the application of the above-mentioned nucleic acid aptamers or the above-mentioned products in the detection of ricin and / or absinthecin.

[0015] This invention provides the application of the above-mentioned nucleic acid aptamers in the preparation of antidotes, wherein the antidotes target toxins including ricin and / or absinthecin.

[0016] The present invention provides an antidote comprising the aforementioned nucleic acid aptamer; the toxins targeted by the antidote include ricin and / or absinthecin.

[0017] This invention provides the application of the above-mentioned nucleic acid aptamers or the above-mentioned antidotes in the preparation of antidote drugs, wherein the toxins targeted by the antidote include ricin and / or absinthecin.

[0018] The present invention provides an antidote drug comprising the aforementioned nucleic acid aptamer; the toxins targeted by the antidote include ricin and / or absinthecin.

[0019] This invention provides the application of the above-mentioned nucleic acid aptamers or the above-mentioned antidotes in the preparation of products for preventing poisoning, wherein the poisons include ricin and / or absinthecin.

[0020] The present invention discloses the following technical effects:

[0021] This invention, based on the toxic damage mechanisms of ricin and abrinogen, adopts a rational drug design approach and employs a nucleic acid chemical modification strategy to design nucleic acid aptamers with a stable "stem-loop" conformation. The core sequence design and modification strategies include, but are not limited to: locking nucleic acid modification of the conserved "GAGA" region; stable conformation design (multiple CG units) of the "stem" region on both sides of the "loop"; thiolation modification of the sequence backbone; and modification of chemical functional groups at the sequence ends (such as cholesterol and biotin). Based on the molecular-level activity evaluation and screening methods previously established by the research group, HPLC was used to evaluate the inhibitory activity of the aptamers using released adenine as a quantitative method. A Besa2B cell exposure model was established, and the toxin inhibitory ability of the above aptamers was evaluated using the CCK8 assay with cell survival rate as an indicator. A poisoning model of ICR female mice (intraperitoneal injection) was established, and the detoxification activity of the above aptamers was evaluated using mouse survival rate as the core indicator. The optimized nucleic acid aptamers obtained based on this method system exhibit good in vitro and in vivo pharmacodynamic activity, achieving significant animal protection rates and prolonged survival time at lethal doses.

[0022] The nucleic acid aptamers or their derivatives provided by this invention are designed and synthesized based on the toxic damage mechanism of ricin and abrin toxin and using a nucleic acid chemical modification strategy. Therefore, their targets are directly the toxic active sites of ricin and abrin toxin, and the binding affinity between the two is strong and the specificity is high.

[0023] The detoxifying agents containing the nucleic acid aptamers provided by this invention have high potency, simple structure, good stability, are easy to chemically synthesize and mass-produce, and have controllable costs. Their in vivo nuclease resistance and half-life can be further enhanced through modification (such as 3'-terminal cholesterol or thiophosphate backbone modification).

[0024] The nucleic acid aptamer antidote provided by this invention has significant advantages in the treatment of ricin and arisaema toxin poisoning: it can be used for both pre-exposure prevention and post-exposure emergency treatment, and has a wide range of applications.

[0025] In addition to serving as an antidote, the nucleic acid aptamer provided by the invention can also be used as a recognition element to develop rapid and sensitive ricin toxin detection test strips or biosensors for public safety monitoring. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The results of the molecular-level evaluation of the inhibitory activity of different nucleic acid aptamers against ricin;

[0028] Figure 2 The nucleic acid aptamer ZT-Apt-15-7 targets ricin EC at the molecular level. 50 Value test results;

[0029] Figure 3 The results of the evaluation of the inhibitory activity of nucleic acid aptamer ZT-Apt-15-7 against ricin at the cellular level;

[0030] Figure 4 Construction of a mouse ricin exposure model;

[0031] Figure 5 The results of the evaluation of the inhibitory activity of nucleic acid aptamer ZT-Apt-15-7 against ricin at the animal level;

[0032] Figure 6 The results of a study on the preventive activity of nucleic acid aptamer ZT-Apt-15-7 against lethal doses of ricin poisoning;

[0033] Figure 7 The results of a study on the therapeutic activity of the nucleic acid aptamer ZT-Apt-15-7 against lethal doses of ricin poisoning;

[0034] Figure 8 The results of the safety evaluation of the nucleic acid aptamer ZT-Apt-15-7 at the cellular level;

[0035] Figure 9 Preliminary safety evaluation results of nucleic acid aptamer ZT-Apt-15-7 at the animal level;

[0036] Figure 10 The results of the molecular-level evaluation of the inhibitory activity of different nucleic acid aptamers against abrinogen toxin;

[0037] Figure 11 The results of the EC50 value assay for the nucleic acid aptamer ZT-Apt-15-7 against abrinogen toxin at the molecular level;

[0038] Figure 12 The results of the evaluation of the inhibitory activity of nucleic acid aptamer ZT-Apt-15-7 against abrinogen toxin at the cellular level;

[0039] Figure 13 Construction of a mouse model of abrinogen exposure;

[0040] Figure 14 The results of a study on the preventive activity of nucleic acid aptamer ZT-Apt-15-7 against lethal doses of abrinogen toxin poisoning;

[0041] Figure 15 The results of a study on the therapeutic activity of the nucleic acid aptamer ZT-Apt-15-7 against lethal doses of abrinogen toxin poisoning. Detailed Implementation

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0045] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0046] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0047] Unless otherwise specified in the following examples, the procedures should be followed according to standard conditions or the manufacturer's recommendations. Raw materials whose manufacturers are not specified are all commercially available products.

[0048] While many of the materials and methods of operation used in the following embodiments are well known in the art, the present invention is still described in as much detail as possible herein. It will be apparent to those skilled in the art that, unless otherwise stated, the materials and methods of operation used in the following embodiments are well known in the art.

[0049] The nucleic acid aptamers (SEQ ID NO.1-SEQ ID NO.9), nucleic acid substrate rA12N (SEQ ID NO.10), and nucleic acid substrate Risub15 (SEQ ID NO.11) used in the embodiments of this invention were synthesized by Shanghai Bioengineering Co., Ltd. Ricin and arisaema toxin were purchased from Beijing Hanpu Pharmaceutical Biotechnology Research Institute. Besa2b cells were purchased from Pronosei. DMEM culture medium, serum, PBS solution, etc., were purchased from Beijing Omega Biotechnology Co., Ltd. CCK8 was purchased from Biosharp. Citric acid, acetic acid, triethylamine, and other chemical reagents were purchased from Sinopharm Group. ICR mice were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0050] The nucleotide sequence of ZT-Apt-15-2 is shown in SEQ ID NO.1, specifically: 5'-C*C*C*C*G*A*G*A*G*G*G*G-3'; where * represents phosphate thioester (PS) modification (thio modification);

[0051] The nucleotide sequence of ZT-Apt-15-3 is shown in SEQ ID NO.2, specifically 5'-CCCCGAGAGGGG-3';

[0052] The nucleotide sequence of ZT-Apt-15-4 is shown in SEQ ID NO.3, specifically: 5'-C*C*C*C*G* / i2MOErA / *G*A*G*G*G*G-3'; where * represents phosphate thioester (PS) modification, and i2MOEr represents 2'-O-methoxyethyl modification, that is, 2'-O-methoxyethyl is modified at the 6th base position;

[0053] The nucleotide sequence of ZT-Apt-15-5 is shown in SEQ ID NO.4, specifically: 5'-C*C*C*C*G* / iXNA_A / *G*A*G*G*G*G-3'; where * represents phosphate thioester (PS) modification and iXNA represents LNA modification, that is, there is LNA modification at the 6th base position;

[0054] The nucleotide sequence of ZT-Apt-15-6 is shown in SEQ ID NO.5, specifically: 5'-C*C*C*C*G* / iXNA_A / *G* / iXNA_A / *G*G*G*G-3' Biotin; where * represents phosphate thioester (PS) modification, iXNA represents LNA modification, that is, LNA modification is present at both the 6th and 8th bases, and biotin is modified at the 3' end;

[0055] The nucleotide sequence of ZT-Apt-15-7 is shown in SEQ ID NO.6, specifically: 5'-C*C*C*C*G* / iXNA_A / *G* / iXNA_A / *G*G*G*G-3'; where * represents phosphate thioester (PS) modification, and iXNA represents LNA modification (locked nucleoside modification), meaning that LNA modification is present at both the 6th and 8th base positions;

[0056] The nucleotide sequence of ZT-Apt-15-7-4 is shown in SEQ ID NO.7, specifically: 5'-C*C*C*C*G* / iXNA_A / *G* / iXNA_A / *G*G*G*G-3'-TriGalNAc; where * represents phosphate thioester (PS) modification, iXNA represents LNA modification, that is, LNA modification is present at both the 6th and 8th bases, and trivalent N-acetylgalactosamine is modified at the 3' end;

[0057] The nucleotide sequence of ZT-Apt-15-8 is shown in SEQ ID NO.8, specifically: 5'-C*C*C*C*G*C*G*A*G*G*G*G-3'; where * represents phosphate thioester (PS) modification;

[0058] The nucleotide sequence of ZT-Apt-15-12 is shown in SEQ ID NO.9, specifically: 5'-C*C*C*C*G* / iXNA_A / *G*A*G*G*G*G-3'-Cholesteryl; where * represents phosphate thioester (PS) modification, iXNA represents LNA modification, and cholesterol is modified at the 3' end;

[0059] The nucleotide sequence of the nucleic acid substrate rA12N is shown in SEQ ID NO.10, specifically: 5'-AAAAAAAAAAAN-3', where N is A, T, C or G;

[0060] The nucleotide sequence of the nucleic acid substrate Risub15 is shown in SEQ ID NO.11, specifically: 5'-UCUCCGCGaGAGCGCGGU-3'.

[0061] In this embodiment of the invention, the nucleic acid aptamer solutions were all prepared with sterile water. The ricin toxin solution and abrin toxin solution were prepared into stock solutions with sterile water, which were subsequently diluted with buffer solutions as needed.

[0062] Example 1: Molecular-level activity evaluation and screening of nucleic acid aptamers

[0063] In the experiment, the concentration of the nucleic acid substrate (rA12N for ricin) used was 100 µM, and the concentration of the ricin protein reaction was 320 nM. The prepared buffer solution was: 20 mM potassium citrate, 2 mM EDTA-Na2, pH 4.0. The specific steps are as follows:

[0064] 1) Take 50 µL of buffer (final concentration of potassium citrate 10 mM, EDTA-Na2 1 mM, pH 4.0) into a 1.5 mL EP tube. Add 10 µL of nucleic acid aptamers of different concentrations (final concentration of 2 µM) and 10 µL of ricin (32 nM) to each reaction system. Add sterile water to a total volume of 90 µL, mix well, and incubate in a 37℃ water bath for 30 min.

[0065] 2) After incubation, add 10 µL of nucleic acid substrate rA12N (final concentration 10 µM) and incubate in a 37°C water bath for 30 min or 60 min.

[0066] 3) After incubation, the adenine area was analyzed by high-performance liquid chromatography (HPLC) and compared with the standard adenine concentration curve. Specific HPLC conditions: Mobile phase A was 0.1 M acetic acid-triethylamine buffer solution (pH 7.45), mobile phase B was chromatographic grade acetonitrile, A / B = 95 / 5, flow rate 1 mL / min, injection volume 80 μL, and detection wavelength 260 nm.

[0067] Following the specific method described in Example 1, the inhibitory activity of different nucleic acid aptamers at certain concentrations against ricin depurination was evaluated. The results are shown in [Figure 1]. Figure 1 .

[0068] The results showed that, compared with the substrate control group, nucleic acid aptamers ZT-Apt-15, ZT-Apt-15-7, and ZT-Apt-15-8 could significantly inhibit the release of adenine, indicating that the aptamers could bind to the toxic active site of ricin and significantly inhibit its toxicity, thus verifying the effectiveness of the sequence design and modification in this invention.

[0069] Example 2: Nucleic acid aptamer EC 50 Value determination

[0070] Following the molecular evaluation method of Implementation Case 1, adenine release was tested in each group by setting different aptamer concentration groups. The adenine release was plotted with Log(aptamer concentration) on the x-axis and (1 - adenine release in the aptamer inhibition group / adenine release in the substrate rA12N control group) * 100 on the y-axis. Nonlinear fitting was performed using GraphPad Prism 8 software to obtain the EC50 values. 50 Value. See results. Figure 2ZT-Apt-15-7 for Ricin's EC 50 The value was 18.52 nM, indicating that the inhibitory activity reached the nM concentration level.

[0071] Example 3: Evaluation of Nucleic Acid Aptamer Cellular Activity

[0072] BEAS-2B cells (Pronosai, CL-0101) were cultured and seeded into 96-well plates at a density of 30,000 cells / mL, with a volume of 100 μL. A 100 nM aptamer solution was added, and the cells were incubated for 12 h. Ricin toxin solution was added to the cultured cells to a final concentration of 1 nM. A blank DMEM medium was used as a normal control group, and the cells were incubated for another 12 h. Then, 10 μL of CCK8 staining agent (BioSharp, Cat. No: BS350C) was added to the incubated cells, and staining was performed for 2 h. The OD value at 450 nm was read using a microplate reader, and the relative cell proliferation rate was calculated. Results are shown below. Figure 3 The results showed that, compared with the ricin-treated control group, ZT-Apt-15-7 and sequences with different terminal modifications all exhibited higher cell viability, indicating that at this concentration, ZT-Apt-15-7 and its derivatives have good preventive and therapeutic effects against ricin.

[0073] Example 4: Construction of a mouse model of ricin exposure

[0074] Female ICR mice (18-21g) were randomly divided into groups of 10 mice each. Different doses of ricin solution (20 μg / kg, 40 μg / kg, 60 μg / kg, 80 μg / kg, or 100 μg / kg) were administered intraperitoneally. A mouse poisoning model was established using the survival rate of mice in each group as the core indicator. Mice mortality was observed and recorded over 4-5 days. The Bliss method was used to fit the data between the ricin dosage and survival rate. The results are shown below. Figure 4 .

[0075] The results showed that mice in different ricin-exposed groups died with prolonged observation time; the higher the dose of ricin, the higher the mortality rate, indicating that the mouse ricin exposure model was successfully established. The LD50 of ricin was calculated using the Bliss method. 50 The value was 33.3 μg / kg, LD50 95 The value was 81.3 μg / kg, which provides data support for the subsequent pharmacodynamic evaluation of the nucleic acid aptamer antidote at the animal level.

[0076] Example 5: Evaluation of the antitoxic activity of nucleic acid aptamers at the animal level (1 LD50)50 (at the dose of toxicity)

[0077] Female ICR mice (18-21 g) were randomly divided into four groups of eight mice each. An equal volume of nucleic acid aptamer solution (concentrations: 2 nM, 10 nM, or 50 nM) and ricin solution (dose: 70 μg / kg) was injected intraperitoneally into the mice. The in vivo antitoxic activity of the nucleic acid aptamer was evaluated using the survival rate within 7 days post-administration. Results are shown below. Figure 5 .

[0078] The results showed that, starting from day 4 post-administration, the survival rate of mice in the ZT-Apt-15-7 experimental groups at different concentrations was significantly higher than that in the Ricin-treated group. On day 7 post-administration, the survival rate of the ZT-Apt-15-7 group (2 nM concentration) was 62.5%, significantly higher than that of the Ricin-treated group (survival rate of only 12.5%), demonstrating good anti-ricin activity at the median lethal dose. This result further validates the high inhibitory activity of the aptamer, demonstrating its ability to maintain good binding and inhibitory activity with ricin even in the complex in vivo environment.

[0079] Example 6: Evaluation of antitoxic activity of nucleic acid aptamers at the animal level (1 LD50) 95 (Prophylactic administration at lethal dose)

[0080] Female ICR mice (18-21 g) were randomly divided into 5 groups of 10 mice each. Different concentrations of aptamer solutions (50 nM, 250 nM, or 1.25 μM) were injected into the mice via the tail vein. The ricin-treated group received an equal volume of PBS solution. Ten minutes later, ricin solution (81 μg / kg) was injected intraperitoneally into all groups. Mouse survival rate was the core indicator, and the survival status of mice in each group was observed over time. Results are shown below. Figure 6 .

[0081] The results showed that all mice in the ricin-treated model group died 32 hours after administration of the lethal dose of ricin. The survival rate of mice in the ZT-Apt-15-7 prevention groups at different concentrations was greater than that in the ricin-treated model group, which prolonged the survival time of mice at the lethal dose, indicating that ZT-Apt-15-7 has a good preventive effect against ricin poisoning.

[0082] Example 7: Evaluation of antitoxic activity of nucleic acid aptamers at the animal level (1 LD50) 95 (Treatment of poisoning at lethal doses)

[0083] Female ICR mice (18-21 g) were randomly divided into 5 groups of 10 mice each. Ricin toxin solution (81 μg / kg) was injected intraperitoneally into all groups. Ten minutes later, different concentrations of aptamer solutions (50 nM, 250 nM, or 1.25 μM) were injected intravenously into the mice. The ricin-treated group received an equal volume of PBS solution. Mouse survival rate was the core indicator, and the survival status of each group was observed over time. Results are shown below. Figure 7 .

[0084] The results showed that the survival rate of mice in the ZT-Apt-15-7 treatment groups at different concentrations was greater than that in the Ricin poisoning model group, prolonging the survival time of mice at the lethal dose. All mice in the poisoning model group died 32 hours after administration, while the protection rate of the poisoning treatment group (ZT-Apt-15-7: 50 nM) reached more than 50% at 32 hours after administration, indicating that ZT-Apt-15-7 has good detoxification activity against ricin poisoning.

[0085] Example 8: Safety evaluation of nucleic acid aptamers at the cellular level

[0086] BEAS-2B cells (Pronosai, CL-0101) were cultured and seeded into 96-well plates at a density of 30,000 cells / mL, with a volume of 100 μL. A 100 nM aptamer solution was added, and the cells were incubated for 12 h. Different concentrations of ZT-Apt-15-7 solution (15 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM) were added to the cultured cells, with blank DMEM medium used as the normal group. The cells were incubated for 48 h. Then, 10 μL of CCK8 staining agent (BioSharp, Cat. No: BS350C) was added to the incubated cells, and staining was performed for 2 h. The OD value at 450 nm was read using a microplate reader, and the relative cell proliferation rate was calculated. The results are shown below. Figure 8 .

[0087] The results showed that there was no significant difference in the relative cell proliferation rate between the groups and the normal group, indicating that different concentrations of ZT-Apt-15-7 had no significant effect on cell proliferation, demonstrating the good biocompatibility of this aptamer at the cellular level.

[0088] Example 9: Animal-level safety evaluation of nucleic acid aptamers

[0089] Female ICR mice (18-21 g) were randomly divided into 5 groups, with 10 mice in each group. Different concentrations of ZT-Apt-15-7 solution (15 μM, 10 μM, 1 μM, and 0.1 μM) were injected into the mice via the tail vein, and were designated as 15-7 (15 μM), 15-7 (10 μM), 15-7 (1 μM), and 15-7 (0.1 μM), respectively. Mice in the control group received an equal volume of PBS solution via tail vein injection, which was recorded as a blank. The condition of the mice in each group was observed over time, and changes in mouse weight were recorded. Results are shown below. Figure 9 .

[0090] The results showed that no mice died during the 14-day observation period, and their activity levels were not significantly different from those of normal mice; their body weight gradually increased over time, with no significant difference compared to the normal group. These results indicate that ZT-Apt-15-7 has good biocompatibility under these experimental conditions.

[0091] Example 10: Molecular-level activity evaluation and screening of nucleic acid aptamers

[0092] In the experiment, the reaction concentration of the nucleic acid substrate (Risub15 for abrinogen toxin) was 100 µM, and the reaction concentration of the abrinogen toxin protein was 160 nM. The prepared buffer solution was: 20 mM potassium citrate, 2 mM EDTA-Na2, pH 4.0. The specific steps are as follows:

[0093] 1) Take 50 µL of buffer (final concentration of potassium citrate 10 mM, EDTA-Na2 1 mM, pH 4.0) into a 1.5 mL EP tube. Add 10 µL of nucleic acid aptamers of different concentrations (final concentration of 2 µM) and 10 µL of abrinogen toxin (32 nM) to each reaction system. Add sterile water to a total volume of 90 µL, mix well, and incubate in a 37℃ water bath for 30 min.

[0094] 2) After incubation, add 10 µL of substrate Risub15 (final concentration 10 µM) and incubate in a 37°C water bath for 30 min or 60 min.

[0095] 3) After incubation, the adenine area was analyzed by high-performance liquid chromatography (HPLC) and compared with the standard adenine concentration curve. Specific HPLC conditions: Mobile phase A was 0.1 M acetic acid-triethylamine buffer solution (pH 7.45), mobile phase B was chromatographic grade acetonitrile, A / B = 95 / 5, flow rate 1 mL / min, injection volume 80 μL, and detection wavelength 260 nm.

[0096] Following the specific method described in Example 1, the inhibitory activity of different concentrations of nucleic acid aptamers on the depurination of abrinogen was evaluated. The results are shown in [Figure 1].Figure 10 .

[0097] The results showed that, compared with the substrate control group, nucleic acid aptamers ZT-Apt-15-2, ZT-Apt-15-7, and ZT-Apt-15-8 could significantly inhibit the release of adenine, indicating that the aptamers could bind to the toxic active site of abrinogen and significantly inhibit its toxicity, thus verifying the effectiveness of the sequence design and modification in this invention.

[0098] Example 11: Nucleic Acid Aptamer EC 50 Value determination

[0099] Following the molecular evaluation method of Implementation Case 10, adenine release was tested in each group by setting different aptamer concentration groups. The adenine release was plotted with Log(aptamer concentration) on the x-axis and (1 - adenine release in the aptamer inhibition group / adenine release in the substrate Risub15 control group) * 100 on the y-axis. Nonlinear fitting was performed using GraphPad Prism 8 software to obtain the EC50 values. 50 Value. See results. Figure 11 ZT-Apt-15-7 for Abrin's EC 50 The value was 84.41 nM, indicating that the inhibitory activity reached the nM concentration level.

[0100] Example 12: Evaluation of Nucleic Acid Aptamer Cellular Activity

[0101] BEAS-2B cells (Pronosai, CL-0101) were cultured and seeded into 96-well plates at a density of 30,000 cells / mL, with a volume of 100 μL. A 100 nM aptamer solution was added, and the cells were incubated for 12 h. Then, a final concentration of 1 nM abrin toxin solution was added to the cultured cells. Blank DMEM medium was used as the normal group, and the treatment without aptamer solution was used as the abrin-treated control group. Both groups were incubated for 12 h. After incubation, 10 μL of CCK8 staining agent (BioSharp, Cat. No: BS350C) was added to the cells, and staining was performed for 2 h. The OD value at 450 nm was read using a microplate reader, and the relative cell proliferation rate was calculated. Results are shown below. Figure 12 The results showed that, compared with the Abrin-treated control group, ZT-Apt-15-7 and sequences with different terminal modifications exhibited higher cell viability, indicating that at this concentration, ZT-Apt-15-7 and its derivatives have good preventive and therapeutic effects against abrin toxin.

[0102] Example 13: Construction of a mouse model of abrinogen exposure

[0103] Female ICR mice (18-21g) were randomly divided into groups of 10 mice each. Different doses of abrinogen toxin solution (90 μg / kg, 110 μg / kg, 130 μg / kg, 150 μg / kg, or 170 μg / kg) were administered intraperitoneally. A mouse poisoning model was established using the survival rate of each group as the core indicator. Mice mortality was observed and recorded over 4-5 days. The Bliss method was used to fit the data between the toxin dosage and survival rate. The results are shown below. Figure 13 .

[0104] The results showed that mice in different concentrations of absinthecin died with prolonged observation time; the higher the dose of absinthecin, the higher the mortality rate, indicating that the mouse poisoning model was successfully established. The LD50 of absinthecin was calculated using the Bliss method. 50 The value was 97.1 μg / kg, LD50 95 The value was 147.2 μg / kg, which provides data support for the subsequent pharmacodynamic evaluation of the nucleic acid aptamer antidote at the animal level.

[0105] Example 14: Evaluation of the antitoxic activity of nucleic acid aptamers at the animal level (1 LD50) 95 (Prophylactic administration at lethal dose)

[0106] Female ICR mice (18-21 g) were randomly divided into 5 groups of 10 mice each. Different concentrations of nucleic acid aptamer solutions (10 nM, 250 nM, or 1.25 μM) were injected into the mice via the tail vein. Normal mice served as controls, while the Abrin-treated group received an equal volume of PBS solution. Ten minutes later, abrin toxin solution (147 μg / kg) was injected intraperitoneally into all groups. Mouse survival rate was the primary indicator, and the survival status of each group was observed over time. Results are shown below. Figure 14 .

[0107] The results showed that all mice in the Abrin-treated model group died 48 hours after administration. The survival rate of mice in the ZT-Apt-15-7 prevention groups at different concentrations was greater than that in the Abrin-treated model group, which prolonged the survival time of mice at the lethal dose. This indicates that ZT-Apt-15-7 has a good preventive effect against Abrin poisoning.

[0108] Example 15: Evaluation of antitoxic activity of nucleic acid aptamers at the animal level (1 LD50) 95 (Treatment of poisoning at lethal doses)

[0109] Female ICR mice (18-21 g) were randomly divided into 5 groups of 10 mice each. Abrin toxin solution (dose 147 μg / kg) was injected intraperitoneally into all groups. Ten minutes later, different concentrations of aptamer solutions (100 nM, 250 nM, 1.25 μM, 5 μM, or 15 μM) were injected intravenously into the mice. Normal mice served as controls, while the Abrin-treated group received an equal volume of PBS solution. Mouse survival rate was the primary indicator, and the survival status of each group was observed over time. Results are shown below. Figure 15 .

[0110] The results showed that the survival rate of mice treated with different concentrations of ZT-Apt-15-7 was greater than that of mice in the Abrin poisoning model group, prolonging the survival time of mice at the lethal dose. At the lethal dose of Abrin, all mice in the poisoning model group died 48 h after administration, while the protection rate of the poisoning treatment groups (10 nM, 5 μM, 15 μM) was over 50%, indicating that ZT-Apt-15-7 has good detoxification activity against abrin poisoning.

[0111] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A nucleic acid aptamer targeting ricin and / or absinthecin, characterized in that, The nucleotide sequence of the nucleic acid aptamer is shown as any one or more sequences in SEQ ID NO.1-SEQ ID NO.

9.

2. The use of the nucleic acid aptamer according to claim 1 in the preparation of products for detecting ricin and / or absinthecin.

3. The application according to claim 2, characterized in that, The products include reagents, reagent kits, and chips.

4. A product for detecting ricin and / or abrinogen, characterized in that, The product includes the nucleic acid aptamer as described in claim 1.

5. The use of the nucleic acid aptamer of claim 1 or the product of claim 4 in the detection of ricin and / or absinthecin.

6. The application of the nucleic acid aptamer according to claim 1 in the preparation of an antidote, characterized in that, The detoxification targets toxins including ricin and / or absinin.

7. An antidote, characterized in that, The antidote includes the nucleic acid aptamer as described in claim 1; the toxins targeted by the antidote include ricin and / or absinthecin.

8. The use of the nucleic acid aptamer of claim 1 or the antidote of claim 7 in the preparation of an antidote drug, characterized in that, The detoxification targets toxins including ricin and / or absinin.

9. An antidote, characterized in that, The detoxifying agent includes the nucleic acid aptamer as described in claim 1; the toxins targeted by the detoxification include ricin and / or absinthecin.

10. The use of the nucleic acid aptamer of claim 1 or the antidote of claim 7 in the preparation of products for preventing poisoning, characterized in that, The toxins include ricin and / or absinin.

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