Artificial nucleic acid aptamer and medical application thereof

Abstract

The invention relates to a nucleic acid aptamer DNA sequence as shown in SEQ ID NO.1 or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing the nucleic acid aptamer sequence or the pharmaceutically acceptable salt thereof as an active ingredient. The invention also discloses application of the nucleic acid aptamer sequence or the pharmaceutically acceptable salt thereof in preparation of antidote drugs used as ricin and / or abrus precatorius toxin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a kind of artificially synthesized single-chain NDA aptamer sequence capable of specifically binding and inhibiting ricin (Ricin) and abrin (Abrin), a composition containing the aptamer, and its application in preparing ricin and abrin antidote. BACKGROUND

[0002] Ricin (Ricin) is a type II ribosome inactivating protein (RIP) extracted from castor seeds, which is connected by two chains of A and B through disulfide bond. B chain binds to cell surface galactose receptor and mediates endocytosis; A chain has RNA N-glycosidase activity, which can catalyze the depurination of eukaryotic ribosome 28S rRNA, leading to protein synthesis inhibition and cell death. Its half lethal dose (LD50) for humans is extremely low, about 0.1-1.0 μg / kg, and there is no specific antidote.

[0003] Abrin (Abrin) has a highly similar structure to ricin and also belongs to type II RIP, which is derived from Abrus precatorius seeds. It is more toxic, with an LD50 of about 0.1 μg / kg, and its mechanism of action is similar to that of ricin, which leads to multiple organ failure by inhibiting protein synthesis. There is also a lack of specific antidote.

[0004] Current strategies for combating ricin and abrin poisoning mainly include small molecule inhibitors and neutralizing antibodies. Small molecule inhibitors generally have weak affinity and poor activity; antibodies are effective in animal models, but large molecule antibody proteins cannot enter cells, have poor antidote effect after poisoning, and have limitations such as high production cost, poor stability, and immunogenicity risk.

[0005] Aptamer is a single-stranded DNA or RNA molecule selected by SELEX technology, which has high affinity, high specificity, can enter cells, is easy to synthesize and modify, has low immunogenicity, and is known as "chemical antibody". However, the natural sequence aptamer obtained by SELEX screening has a long sequence and poor stability, and cannot be used as a nucleic acid drug, so it needs to be chemically modified to obtain artificial nucleic acid aptamer sequence. There are some literatures reporting the use of aptamer for ricin detection, but there are few studies on antidote drugs, and no aptamer with antidote effect on ricin and abrin has been reported, and there are problems such as low cell uptake rate, poor in vivo plasma stability, and short half-life.

[0006] Therefore, it is of great medical and public safety value to develop a nucleic acid aptamer antidote candidate drug that can simultaneously resist ricin and abrin. SUMMARY

[0007] (I) OBJECTIVE

[0008] The present application aims to provide a nucleic acid aptamer and its derivatives with high affinity and high inhibitory activity to ricin and abrin.

[0009] Further, the present application provides a sequence design, modification optimization and activity screening method of the nucleic acid aptamer.

[0010] Further, the present application provides the use of the nucleic acid aptamer in the preparation of a medicament for preventing or treating ricin and abrin poisoning.

[0011] Further, the present application provides a pharmaceutical composition comprising the nucleic acid aptamer.

[0012] Further, the present application provides the use of the nucleic acid aptamer in the preparation of a ricin and abrin detection reagent or kit.

[0013] (II) TECHNICAL SCHEME

[0014] The present application provides an artificially synthesized nucleic acid aptamer RIN62 as shown in SEQ ID NO. 1, which can specifically bind to ricin and abrin and inhibit the toxic activity thereof.

[0015] The nucleic acid aptamer SEQ ID NO. 1 is a single-stranded DNA sequence, which can be optionally chemically modified to enhance stability, affinity or in vivo half-life, including but not limited to 2'-fluoro modification, 2'-oxymethyl modification, phosphorothioate backbone modification, PEGylation modification, etc.

[0016] The aptamer sequence SEQ ID NO. 1 is based on the mechanism of ricin and abrin poisoning, simulates the natural RNA sequence of the target of the toxin, and uses the key technical means of nucleic acid chemical modification to design and synthesize an artificial single-stranded DNA sequence, which can realize high specificity binding to toxin protein and competitively inhibit the action of toxin on the target.

[0017] The aptamer SEQ ID NO. 1 can be used alone or in combination with a pharmaceutically acceptable carrier to form a pharmaceutical composition for preventing or treating ricin and / or abrin poisoning.

[0018] The aptamer SEQ ID NO. 1 can also be used to prepare a toxin detection kit, including but not limited to ELISA, colloidal gold test strip, biosensor, etc.

[0019] SEQ ID NO. 1 (RIN62):

[0020] TCTGGGY 1GACCAGT

[0021] wherein:

[0022] Y 1 is .

[0023] The present application claims a nucleic acid aptamer represented by SEQ ID NO. 1 or a pharmaceutically acceptable salt thereof,

[0024] SEQ ID NO. 1 (RIN62): TCTGGGY 1 GACCAGT

[0025] wherein:

[0026] Y 1 is .

[0027] In the synthesis of the nucleic acid aptamer represented by SEQ ID NO. 1, the monomer used for the Y 1 residue is .

[0028] The present application also claims a pharmaceutical composition containing the nucleic acid aptamer represented by SEQ ID NO. 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable excipient or carrier.

[0029] In certain embodiments, the pharmaceutical composition is a solution, a tablet, a capsule, or an injection.

[0030] The present application also claims the use of the nucleic acid aptamer represented by SEQ ID NO. 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the nucleic acid aptamer represented by SEQ ID NO. 1 or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a medicine as a detoxifying agent for ricin and / or abrin or in the preparation of a medicine for preventing or treating ricin and / or abrin poisoning.

[0031] The present application also claims a kit for the detection of ricin and / or abrin, comprising the nucleic acid aptamer represented by SEQ ID NO. 1 or a pharmaceutically acceptable salt thereof.

[0032] The present application also claims the use of the nucleic acid aptamer represented by SEQ ID NO. 1 or a pharmaceutically acceptable salt thereof in the detection of ricin and / or abrin (in vivo or in vitro).

[0033] The present application also claims the use of the aptamer or its pharmaceutically acceptable salt as shown in SEQ ID NO. 1 for preparing a kit for detecting ricin and / or abrin. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Standard curve for adenine;

[0035] Figure 2 Evaluation results of the inhibitory activity of the aptamer RIN62 against ricin at the molecular level;

[0036] Figure 3 EC value test results of the aptamer RIN62 against ricin and abrin at the molecular level; 50

[0037] Figure 4 Evaluation results of the inhibitory activity of the aptamer RIN62 against ricin and abrin at the cellular level;

[0038] Figure 5 Construction of a mouse ricin poisoning model;

[0039] Figure 6 Preventive activity results of the aptamer RIN62 against lethal dose ricin poisoning;

[0040] Figure 7 Therapeutic activity results of the aptamer RIN62 against lethal dose ricin poisoning;

[0041] Figure 8 Construction of a mouse abrin poisoning model;

[0042] Figure 9 Preventive activity results of the aptamer RIN62 against lethal dose abrin poisoning;

[0043] Figure 10 Therapeutic activity results of the aptamer RIN62 against lethal dose abrin poisoning;

[0044] Figure 11 and Figure 12 Animal level safety evaluation results of the aptamer RIN62. DETAILED DESCRIPTION

[0045] ​The essential content of the present application will be further illustrated by specific examples of the present application. It should be understood that the following examples are only used to illustrate the present application, but not to limit the protection scope of the present application. The specific conditions are not indicated in the following examples, which are carried out according to the conventional conditions or the manufacturer's recommendations. The raw materials used are conventional products that can be obtained by purchase.

[0046] Although many materials and operation methods used in the following examples are well known in the art, the present application still describes them as much as possible. It is clear to those skilled in the art that the materials and operation methods used in the following examples are well known in the art if not specifically described.

[0047] The nucleic acid aptamer RIN62 and nucleic acid substrate rA12N used in the examples of the present application were synthesized by Shanghai Bioengineering Co., Ltd., ricin and abrin were purchased from Beijing Hansu Pharmaceutical and Biological Research Institute, Beas-2b cells were purchased from Pnuo Sai, DMEM medium was purchased from Thermo Fisher (Suzhou) Instrument Co., Ltd., serum was purchased from Beijing Omiya Biological Technology Co., Ltd., PBS solution was purchased from Saver Biological, CCK-8 was purchased from biosharp company; citric acid monohydrate, acetic acid, triethylamine and other chemical reagents were purchased from National Pharmaceutical Group. ICR mice were purchased from Spire Biotechnology (Beijing) Co., Ltd.

[0048] In the examples of the present application, the nucleic acid aptamer solution was prepared with sterilized water. The ricin and abrin solutions were prepared with sterilized water as stock solutions, and then diluted with buffer solution as needed.

[0049] Example 1: Synthesis of RIN62 artificial aptamer sequence

[0050] Using a full-automatic DNA solid-phase synthesizer, the aptamer sequence was synthesized by the phosphoramidite method using natural nucleoside phosphoramidite monomers and modified structure unit phosphoramidite monomers (Y 1 ), and the sequence was fully modified with thiol. Purified by HPLC, the purity was >95.0%. The molecular weight was confirmed by ESI-MS. As shown in Table 1, the difference between the measured value and the calculated value was within the instrument error range, indicating that the molecular weight of the synthesized aptamer sequence was correct.

[0051] Table 1 Sequence molecular weight of RIN62

[0052]

[0053] Example 2: Construction of adenine standard curve

[0054] The adenine standard solution of different concentrations was prepared using a buffer (20 mM potassium citrate, 2 mM EDTA-Na2, pH 4.0) and analyzed by area analysis under the same conditions using high performance liquid chromatography to make a standard curve between the mass of adenine and the area, and the results are shown in Table 1. Figure 1 .

[0055] Example 3: Evaluation of the inhibition of depurination activity at the molecular level by RIN62

[0056] The reaction concentration of the nucleic acid substrate (rA12N for ricin and Risub15 for abrin) in the experiment was 100 µM, the reaction concentration of the ricin protein was 320 nM, and the reaction concentration of the abrin protein was 160 nM. The prepared buffer was: 20 mM potassium citrate, 2 mM EDTA-Na2, pH 4.0.

[0057] The specific steps are as follows: 50 µL of buffer (final concentration of potassium citrate 10 mM, EDTA-Na2 1 mM, pH 4.0) was taken in a 1.5 mL EP tube, 10 µL of nucleic acid aptamer inhibitor (final concentration of 2 µM) of different concentrations and 10 µL of ricin (32 nM) were added to each reaction system, sterilized water was added to a total volume of 90 µL, mixed, and incubated in a 37°C water bath for 30 min. After incubation, 10 µL of substrate rA12N (final concentration of 10 µM) was added, and incubated in a 37°C water bath for 30 min or 60 min. After incubation, the adenine area was analyzed by high performance liquid chromatography, and compared with the standard adenine concentration curve. The specific conditions of HPLC are as follows: mobile phase A is 0.1 M acetic acid-triethylamine buffer salt solution (pH 7.45), mobile phase B is chromatographic grade acetonitrile, A / B = 95 / 5, flow rate 1 mL / min, injection volume is 80 μL, and detection wavelength is 260 nm.

[0058] According to the above method, the inhibition activity of RIN62 on ricin and abrin depurination was evaluated, and the results are shown in Table 2. Figure 2 .

[0059] The results show that, compared with the substrate control group, the nucleic acid aptamer RIN62 can significantly inhibit the release of adenine, indicating that RIN62 can bind to the toxic active site of ricin and abrin and significantly inhibit the toxicity thereof, verifying the effectiveness of the sequence design and modification in the present application.

[0060] Example 4: Determination of the EC 50 value of RIN62

[0061] According to the evaluation method of Example 3, by setting different RIN62 concentration groups, the adenine release amount of each group was tested, taking Log (aptamer concentration) as the horizontal coordinate, taking (1-adenine release amount of aptamer inhibition group / adenine release amount of substrate (rA12N or Risub15) control group) x 100 as the vertical coordinate, using software GraphPad Prism 8 for non-linear fitting, and obtaining EC 50 values. The results are shown in Figure 3 . The EC 50 value of RIN62 for Ricin is 75.02 nM, and the EC 50 value for Abrin is 322.1 nM.

[0062] Example 5: Evaluation of RIN62 cell level activity

[0063] Beas-2b cells (Pulno Sai, CL-0101) were cultured, and the logarithmic growth phase was used for plating. The cells were cultured in a 96-well plate at a density of 60,000 cells / mL and a volume of 100 μL, and incubated in a cell incubator for 10 h.

[0064] 1) RIN62 was diluted with sterile water containing 1 mM magnesium ions and 15 nM Ricin to different concentrations and incubated in a 37°C water bath for 6 h. A 0.1 nM Ricin control was set during the incubation process. After 6 h of incubation, it was diluted 10 times with DMEM. 12 hours after administration, staining (10 μl of CCK-8 staining solution was added to each well), staining for 3 hours, and the OD value at 450 nm wavelength was read by a microplate reader to calculate the relative proliferation rate of the cells.

[0065] 2) RIN62 was diluted with sterile water containing 1 mM magnesium ions and 0.5 nM Abrin to different concentrations and incubated in a 37°C water bath for 6 h. A 0.05 nM Abrin control was set during the incubation process. After 6 h of incubation, it was diluted 10 times with DMEM. 12 hours after administration, staining (10 μl of CCK-8 staining solution was added to each well), staining for 3 hours, and the OD value at 450 nm wavelength was read by a microplate reader to calculate the relative proliferation rate of the cells. The results are shown in Figure 4 .

[0066] Figure 4 The results show that compared with the Ricin and Abrin poisoning control groups, RIN62 shows a higher cell survival rate, indicating that at this concentration, RIN62 has good preventive and therapeutic effects on ricin and abrin.

[0067] Example 6: Construction of a ricin mouse poisoning model

[0068] ICR female mice (18-21 g) were randomly divided into groups, 10 mice in each group; different doses of ricin solution were injected intraperitoneally (dose: 20 μg / kg, 40 μg / kg, 60 μg / kg, 80 μg / kg, 100 μg / kg), and the survival rate of mice in each group was used as the core index to construct the mouse poisoning model. The death of mice in each group was observed and recorded within 4-5 days, and Bliss method was used to fit the data of poisoning dose and survival rate, and the results are shown in Figure 5 .

[0069] The results show that as the observation time is prolonged, the mice in different concentration poisoning groups die, the larger the ricin poisoning dose, the higher the mortality, which shows that the mouse poisoning model is successfully constructed. According to Bliss method, the LD 50 value of ricin is 33.3 μg / kg, and the LD 95 value is 81.3 μg / kg. This data provides data support for the pharmacodynamic evaluation of nucleic acid aptamer antidote at the animal level.

[0070] Example 7: Evaluation of RIN62 in vivo against ricin activity (1 LD 95 fatal dose, prophylactic administration)

[0071] ICR female mice (19-22 g) were randomly divided into 7 groups, 10 mice in each group; different concentrations of RIN62 solution (concentration: 0.01 µM, 0.05 µM, 0.25 µM, 1.25 µM, 5 µM, 15 µM) were injected into the mice through the tail vein, and the Ricin poisoning group was injected with the same volume of PBS solution; 10 min later, ricin solution (dose: 81 μg / kg) was injected into all groups of mice through the abdominal cavity, and the survival rate of mice was used as the core index. The survival of mice in each group was observed with time, and the results are shown in Figure 6 .

[0072] Figure 6 The results show that under the condition of one lethal dose of Ricin, the mice in the poisoning model group all died 30 h after being given ricin; the survival rate of mice in the RIN62 prophylactic group with different concentrations was higher than that of the Ricin poisoning model group, which prolonged the survival time of mice under the lethal dose, and the survival rate of mice in the 5 μM RIN62 administration group was 75% at 32 h, indicating that RIN62 has good prophylactic effect against ricin poisoning.

[0073] Example 8: Evaluation of RIN62 in vivo against ricin activity (1 LD 95 fatal dose, poisoning after detoxification)

[0074] ICR female mice (19-22 g) were randomly divided into 7 groups, 10 mice in each group; ricin solution (dose: 81 μg / kg) was injected into all groups of mice by intraperitoneal injection; 10 min later, different concentrations of RIN62 solution (concentrations: 0.01 µM, 0.05 µM, 0.25 µM, 1.25 µM, 5 µM, 15 µM) were injected into the mice by tail vein injection, and the Ricin poisoning group was injected with the same volume of PBS solution; the survival rate of mice was used as the core index, and the survival of mice was observed over time, and the results are shown in Figure 7 .

[0075] Figure 7 The results show that the survival rate of mice in different concentrations of RIN62 treatment groups is higher than that of the Ricin poisoning model group, and the survival time of mice under the lethal dose is prolonged; all mice in the poisoning model group died 32 h after administration, while the survival rate of mice in the poisoning treatment group was higher than that of the Ricin poisoning model group, and the survival time of mice under the lethal dose was prolonged, and the survival rate of mice in the 1.25 μM RIN62 administration group was 50% at 32 h, indicating that RIN62 has good detoxification activity for ricin poisoning.

[0076] Example 9: Construction of Abrin Mouse Poisoning Model

[0077] ICR female mice (19-22 g) were randomly divided into groups, 10 mice in each group; different doses of Abrin solution (doses: 20 μg / kg, 40 μg / kg, 60 μg / kg, 80 μg / kg, 100 μg / kg) were administered by intraperitoneal injection, and the survival rate of mice was used as the core index to construct the mouse poisoning model, and the death of mice in each group was observed and recorded within 4-5 days, and Bliss method was used to fit the data of poisoning dose and survival rate, and the results are shown in Figure 8 .

[0078] Figure 8 The results show that with the extension of observation time, mice in different concentration poisoning groups died, the larger the Abrin poisoning dose, the higher the mortality, indicating that the mouse poisoning model was successfully constructed. According to Bliss method, the LD 50 value of Abrin is 97.1 μg / kg, and the LD 95 value is 147.2 μg / kg, which provides data support for the pharmacodynamic evaluation of aptamer antidote at the animal level.

[0079] Example 10: Evaluation of RIN62 Anti-Abrin Activity in Mice (1 LD 95 Lethal dose, prophylactic administration)

[0080] ICR strain female mice (19-22 g), randomly divided into 7 groups, 10 in each group; different concentrations of aptamer solution (dose: 0.05 μM, 0.25 μM, 1.25 μM, 5 μM, 15 μM) were injected into the mice through the tail vein, and the Abrin poisoning group was injected with an equal volume of PBS solution; 10 min later, the Abrin solution (dose 147 μg / kg) was injected into all groups of mice through the abdominal cavity, and the survival rate of mice was observed as the core index, and the survival of mice was observed over time, and the results are shown in Figure 9 .

[0081] Figure 9 The results show that under the lethal dose of Abrin, the mice in the poisoning model group all died after 52 h of administration; the survival rate of mice in the RIN62 prevention group of different concentrations was higher than that of the Abrin poisoning model group, and the survival time of mice under the lethal dose was prolonged, and the survival rate of mice in the 1.25 μM RIN62 administration group was 65% at 52 h, indicating that RIN62 has a good preventive effect on Abrin poisoning.

[0082] Example 11: Evaluation of the activity of RIN62 against Abrin in mice (1 LD 95 Under the lethal dose, detoxification after poisoning

[0083] ICR strain female mice (19-22 g), randomly divided into 7 groups, 10 in each group; the Abrin solution (dose 147 μg / kg) was injected into all groups of mice through the abdominal cavity; 10 min later, different concentrations of RIN62 solution (dose: 0.05 μM, 0.25 μM, 1.25 μM, 5 μM, 15 μM) were injected into the mice through the tail vein, and the Abrin poisoning group was injected with an equal volume of PBS solution; the survival rate of mice was observed as the core index, and the survival of mice was observed over time, and the results are shown in Figure 10 .

[0084] Figure 10 The results show that under the lethal dose of Abrin, the mice in the poisoning model group all died after 52 h of administration; the survival rate of mice in the RIN62 prevention group of different concentrations was higher than that of the Abrin poisoning model group, and the survival time of mice under the lethal dose was prolonged, and the survival rate of mice in the 1.25 μM RIN62 administration group was 65% at 52 h, indicating that RIN62 has a good preventive effect on Abrin poisoning.

[0085] Example 12: Safety evaluation of RIN62 at the cell level

[0086] Beas-2b cells (Pulno Sai, CL-0101) were cultured, and the logarithmic growth phase was used for plating. The cells were cultured in a 96-well plate at a density of 30,000 cells / mL and a volume of 100 μL, and incubated in a cell incubator for 12 h. Different concentrations of RIN62 solution (10 μM, 1 μM, 0.1 μM, 0.01 μM) were added to the cultured cells, and blank DMEM medium was used as a blank control group. The cells were incubated in a cell incubator for 48 h. Then 10 μL of CCK-8 staining agent was added to the incubated cells, and the cells were stained for 2 h. The OD value at 450 nm was read by a microplate reader, and the relative proliferation rate of the cells was calculated. The results are shown in Figure 11 .

[0087] Figure 11 The results show that the relative proliferation rate of the cells in each group has no significant difference compared with the normal group, indicating that different concentrations of RIN62 have no significant effect on cell proliferation, and showing that RIN62 has good safety at the cellular level.

[0088] Example 13: Overall evaluation of RIN62 in mice in vivo

[0089] ICR female mice (19-22 g) were randomly divided into 4 groups, 6 in each group. Different concentrations of RIN62 solution (concentrations: 10 μM, 1 μM, 0.1 μM) were injected into the mice via the tail vein, once a day, for 14 consecutive days. The normal group of mice was given an equal volume of PBS solution by tail vein injection. The state of the mice in each group was observed over time, and the body weight changes of the mice were recorded. The results are shown in Figure 12 .

[0090] Figure 12 The results show that there was no death of mice during the 14-day observation period, and the activity was not significantly different from that of normal mice. The body weight gradually increased over time, and there was no significant difference from the normal group. The results show that RIN62 has good safety under the conditions of this test.

[0091] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details in accordance with all the teachings disclosed herein, and such changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A nucleic acid aptamer represented by SEQ ID NO. 1 or a pharmaceutically acceptable salt thereof, SEQ ID NO. 1: TCTGGGY 1 GACCAGT wherein: Y 1 For .

2. A pharmaceutical composition comprising the nucleic acid aptamer of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable excipient or carrier.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is a solution, a tablet, a capsule or an injection.

4. Use of the nucleic acid aptamer of claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the nucleic acid aptamer of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient for the preparation of a medicament as a detoxifying agent for ricin and / or abrin or for the preparation of a medicament for the prevention or treatment of ricin and / or abrin poisoning.

5. A kit for the detection of ricin and / or abrin, comprising the nucleic acid aptamer of claim 1 or a pharmaceutically acceptable salt thereof.

6. Use of the nucleic acid aptamer of claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a kit for the detection of ricin and / or abrin.

7. Use of the nucleic acid aptamer of claim 1 or a pharmaceutically acceptable salt thereof for the detection of ricin and / or abrin (in vivo or in vitro).

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