Application of malachite green aptamer in cytotoxicity masking

Abstract

The invention discloses an application of a malachite green aptamer in cytotoxicity masking. Firstly, the stability of multiple aptamer sequences in serum and the biological safety of the aptamer sequences are verified, and it is confirmed that the aptamer sequences are suitable for cell level research. Furthermore, through CCK8 detection, cell apoptosis and cycle analysis, mitochondrial membrane potential and ROS level evaluation, the system reveals the relieving effect of the MG aptamer on MG-induced toxicity in HeLa and HEK293T cells. Results show that the high-affinity aptamer can be combined with MG, so that the cell survival rate is remarkably improved, and cell apoptosis, cycle disorder, mitochondrial damage and oxidative stress are reduced. The strategy provides a new path for MG toxicity control, and shows the application prospect of the aptamer in biosafety regulation and control.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of a malachite green aptamer in cytotoxicity masking. Background Art

[0002] Malachite green (MG) is a triphenylmethane dye widely used in aquaculture, textile dyeing, and bio-dyeing. It exhibits excellent antifungal and antiparasitic activity at low concentrations and has long been used as an antimicrobial agent in aquaculture. However, studies have shown that MG is slowly metabolized in animals and exhibits significant cytotoxicity, mutagenicity, and carcinogenicity. Long-term exposure to MG may pose a serious threat to human health and the ecological environment. Consequently, the use of MG has been restricted or banned in many countries and regions. However, in practical applications, illegal use and residues of MG remain widespread, necessitating the development of efficient and safe masking and removal strategies to reduce its toxicity risks.

[0003] Aptamers are a class of specific nucleic acid molecules, obtained through in vitro screening, that bind to target molecules with high affinity and selectivity. Compared to traditional antibodies, aptamers offer advantages such as ease of synthesis, high stability, and controllable modifiability. In recent years, malachite green aptamers have become ideal biorecognition elements for constructing detection platforms due to their extremely high binding affinity and specificity for malachite green. However, research has primarily focused on the application of aptamers in the detection and enrichment of malachite green, while their application in regulating cell behavior is still in its infancy.

[0004] Malachite green aptamers have been applied to mask cytotoxicity. By stabilizing and blocking MG molecules, they significantly reduce MG adsorption and internalization on the cell surface, thereby minimizing its damage to cell membrane structure, mitochondrial function, and oxidative stress. This strategy not only provides a new solution for the safe regulation of MG pollution but also opens new avenues for the application of functional nucleic acids in toxicity control, biobarrier construction, and precision medicine. Therefore, developing a method to effectively mask MG toxicity using malachite green aptamers has important theoretical significance and practical application value. Summary of the Invention

[0005] Based on this, the present invention proposes an aptamer with improved malachite green performance and its application in cytotoxicity masking.

[0006] In one aspect, the present invention provides a malachite green nucleic acid aptamer with improved performance, wherein the aptamer sequence is shown in any one of SEQ ID NOs: 2 to 5.

[0007] The nucleotide sequence of the above-mentioned nucleic acid aptamer is modified and the modified nucleic acid aptamer binds to malachite green, and the modification is selected from at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium and isotopization.

[0008] On the other hand, the present invention provides a conjugate of a malachite green nucleic acid aptamer, wherein the conjugate is a substance for labeling, detection, diagnosis or treatment connected to the nucleotide sequence of the above-mentioned nucleic acid aptamer, and the conjugate of the nucleic acid aptamer after connection with the substance is bound to malachite green, and the substance is a fluorescent marker such as FAM, a radioactive substance, a therapeutic substance, biotin, digoxin, a nanoluminescent material, a small peptide, siRNA and an enzyme label.

[0009] On the other hand, the present invention provides use of the malachite green nucleic acid aptamer shown in SEQ ID NO: 5 in alleviating the cytotoxicity caused by malachite green.

[0010] The aforementioned alleviation of the cytotoxicity caused by malachite green refers to the ability to alleviate the effects of malachite green on the cell cycle or cell apoptosis rate or mitochondrial membrane potential or oxidative stress level.

[0011] The above-mentioned malachite green concentration is 1 μM.

[0012] The concentration of the malachite green nucleic acid aptamer is 2-5 μM.

[0013] On the other hand, the present invention provides a composition for alleviating cytotoxicity caused by malachite green, wherein the composition comprises malachite green nucleic acid aptamers shown in SEQ ID NOs: 2 to 5 as active ingredients.

[0014] Application of the above composition in alleviating cytotoxicity caused by malachite green.

[0015] On the other hand, the present invention provides a preparation for alleviating the cytotoxicity caused by malachite green, characterized in that the preparation contains the malachite green nucleic acid aptamer shown in SEQ ID NO: 2 to 5 as an active ingredient, and the preparation is selected from tablets, capsules, granules, powders, and pills.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. Through structural modification and sequence optimization, the present invention successfully obtained multiple malachite green aptamer variants (such as MC-DA, MC-DA-LG, and CAG-T-ML-S4-TA-AT+1-4L). While maintaining recognition specificity, it achieved a significant increase in fluorescence signal intensity, up to more than 6 times that of the original sequence;

[0018] 2. The nucleic acid aptamer sequence constructed by the present invention exhibits excellent structural stability in a serum environment, maintaining intact bands for 96 hours, significantly outperforming traditional malachite green aptamers. This demonstrates its excellent application stability in complex body fluid environments and is suitable for cellular and in vivo biosensing or intervention studies.

[0019] 3. The aptamer sequence proposed in this invention can form a stable complex with malachite green, masking its cytotoxicity through specific binding, effectively improving cell survival, reducing apoptosis levels, and alleviating adverse reactions such as decreased mitochondrial membrane potential and increased ROS. This is the first systematic verification of the toxicity shielding and detoxification function of the malachite green aptamer at the cellular level;

[0020] 4. The aptamer of the present invention has good biocompatibility and dose-dependent protective effects. It does not induce cytotoxicity at different concentrations and has the ability to recover from malachite green-induced cell cycle disorders, mitochondrial dysfunction and oxidative stress. This shows the broad application prospects of nucleic acid aptamers as biological regulation tools in the field of malachite green sustained release and toxicity regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Sequence fluorescence verification and serum stability assessment. A shows the luminescence performance of different sequences; B shows the 96-hour stability assessment of different sequences in serum.

[0022] Figure 2 Cell viability evaluation of HeLa or HEK293T cells after treatment with different concentrations of MG. A: HeLa cells; B: HEK293T cells.

[0023] Figure 3 Cell viability evaluation of HeLa or HEK293T cells after treatment with different concentrations of the CAG-T-ML-S4-TA-AT+1-4L sequence. A: HeLa cells; B: HEK293T cells.

[0024] Figure 4Evaluation of the detoxification effect of nucleic acid aptamers on MG at the cellular level. A is the cell viability of HeLa cells treated with 1μM MG and different nucleic acid sequences (2μM) using CCK-8; B is the cell viability of HEK293T cells treated with 1μM MG and different nucleic acid sequences (2μM) using CCK-8; C is the cell viability of HeLa cells treated with 1μM MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence using CCK-8; D is the cell viability of HEK293T cells treated with 1μM MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence using CCK-8; E is the cell viability of HeLa cells treated with 1μM MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence Figure 3 is the result of cell apoptosis under treatment with MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence; F is the result of cell cycle under treatment with 1 μM MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence; G is the quantitative result of cell cycle under treatment with 1 μM MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence; H is the quantitative analysis result of cell apoptosis under treatment with 1 μM MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence; I is the quantitative result of mitochondrial membrane potential under treatment with 1 μM MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence; J is the quantitative analysis of ROS levels in HeLa cells under treatment with 1 μM MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence.

[0025] Figure 5 Flow cytometric analysis of apoptosis in HeLa cells after treatment with 1 μM MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence.

[0026] Figure 6 The cell cycle analysis diagram of HeLa cells after treatment with 1 μM MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence.

[0027] Figure 7 Quantitative results of cell cycle analysis of HeLa cells after treatment with 1 μM MG and different concentrations of CAG-T-ML-S4-TA-AT+1-4L sequence. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0029] Example 1. Aptamer performance investigation

[0030] All nucleotide sequences involved in the experiment are shown in Table 1.

[0031] Table 1 Nucleotide sequences involved in the experiment

[0032]

[0033]

[0034] 1. Fluorescence performance

[0035] MC-DA, MC-DA-LG, CAG-T-ML-S4-TA-AT+1, and CAG-T-ML-S4-TA-AT+1-4L are all malachite green aptamer sequences modified from the original sequence. Their fluorescence intensity is greatly enhanced compared to the original sequence. Figure 1 As shown in A, the highest enhancement was more than 6 times.

[0036] 2. Serum stability

[0037] This study aims to explore the potential of various malachite green aptamers in cellular applications. Given the relatively severe cytotoxicity of MG, it is crucial to design a molecular tool that can selectively bind to MG molecules and neutralize their toxic effects. The experiment first evaluated the serum stability of different malachite green aptamer sequences. The original sequence, MC-DA-LG, and CAG-T-ML-S4-TA-AT+1-4L sequences all showed relatively superior stability within 96 hours. Figure 1 As shown in Figure B, the sequence has good serum stability within 96 hours, and obvious residual nucleic acid bands are shown in the agarose gel image, indicating that the aptamer sequence can remain stable under serum conditions for a long time and is suitable for cell-level or in vivo applications.

[0038] 3. Biosafety

[0039] After demonstrating the relatively superior serum stability of the aptamer sequence, the cytotoxicity of MG and the aptamer sequence was further explored. Since MG has been reported to have nephrotoxic and carcinogenic properties, HeLa cells and HEK293T cells were selected as models for evaluating MG cytotoxicity. Figure 2 As shown in A, for HeLa cells, incubation with MG concentrations of 1 μM and below for 24 h can maintain cell viability above 90%. HEK293T cells are slightly sensitive to MG, with 0.5 and 1 μM MG maintaining cell viability at 82.70% and 78.39%, respectively. Figure 2 B). Different concentrations of the CAG-T-ML-S4-TA-AT+1-4L nucleic acid sequence had no significant effect on the viability of HeLa and HEK293T cells, indicating that the aptamer has high biosafety ( Figure 3 ).

[0040] Example 2. Evaluation of the detoxification effect of nucleic acid aptamers on MG at the cellular level

[0041] After confirming the biosafety and dosage of MG and aptamers, the study further systematically evaluated the efficacy of nucleic acid aptamers in masking MG toxicity at the cellular level. MC-DA, MC-DA-LG, CAG-T-ML-S4-TA-AT+1, CAG-T-ML-S4-TA-AT+1-4L sequences were selected as positive treatment sequences, and polyT26 and Scrambled sequences (sequences with the same base composition ratio but different base arrangement order) were used as controls to explore the functionality of specific aptamer sequences. The detoxification efficacy of different aptamer sequences on MG was evaluated by CCK8 experiments. The cell survival rate of the group with no aptamer added and only MG added was 100% by default. Figure 4 As shown in A, in HeLa cells, the addition of pre-bound complexes of different MG aptamer sequences and MG significantly increased cell survival compared to the control group. Among them, the cell survival rate of the MG@CAG-T-ML-S4-TA-AT+1-4L sequence group was the highest, reaching 130.60%. However, no significant effect on cell survival was observed when Poly T26 or Scrambled sequences were introduced, indicating that MG specifically interacts with its aptamer sequence, thereby mediating the toxicity masking and detoxification process of MG at the cellular level. Similar experimental phenomena were also observed in HEK293T ( Figure 4 B). Moreover, in both HeLa and HEK293T cells, this toxicity masking effect increased in a dose-dependent manner with increasing aptamer concentration ( Figure 4 C~D).

[0042] The experiment further evaluated the effect of the aptamer on cell apoptosis. Since the CAG-T-ML-S4-TA-AT+1-4 sequence produced excellent MG toxicity masking efficacy, the subsequent experiments selected 1μM MG and the binding complex of this sequence (low dose: 2μM; high dose: 5μM) as the administration reagent. The effect of the introduction of the aptamer sequence on the MG toxicity masking effect was explored by flow cytometry. Figure 4 E, H and Figure 5 It can be seen that the addition of MG to HeLa cells caused a significant increase in cell apoptosis. After the addition of low and high doses of the aptamer sequence, this increase in apoptosis rate was significantly inhibited, and the cell apoptosis rate was not significantly different from the control group.

[0043] The experiment further evaluated the effect of the aptamer on the cell cycle. MG can disrupt the cell cycle process, leading to cell cycle arrest or abnormal division. By analyzing the changes in the cell cycle, it can be evaluated whether the aptamer sequence can reduce the toxicity of MG. Figure 4 F, G and Figures 6-7 As shown, MG treatment (1 μM) resulted in a significant decrease in the proportion of cells in the G1 / G0 and G2 / M phases, while the proportion of cells in the S phase increased from 24.7% to 33.7%. This suggests that MG can arrest the cell cycle in the S phase, potentially interfering with DNA synthesis. This effect was alleviated by the addition of the aptamer. The cell cycle distribution in the high-dose group (MG@5 μM aptamer sequence) was similar to that in the control group.

[0044] The experiment further evaluated the effect of the aptamer on mitochondrial membrane potential. Apoptosis is a programmed cell death process caused by the activation of metabolic enzymes. During this process, mitochondria undergo morphological and biochemical changes and play a central role in regulating this process. JC-1 staining was used to evaluate whether the aptamer sequence could protect against MG-induced mitochondrial membrane potential dysfunction. Figure 4 As shown in Figure 1, the improvement in the JC-1 fluorescence ratio (green / red) indicates that the CAG-T-ML-S4-TA-AT+1-4L sequence has a certain protective effect against toxicity. Compared with the control group, MG treatment significantly increased the JC-1 fluorescence ratio by 1.51-fold; however, after the addition of the aptamer sequence, this ratio decreased significantly. The high-dose group (5 μM aptamer) showed no significant difference from the control group, indicating that the aptamer can alleviate MG-induced mitochondrial dysfunction.

[0045] The experiment further evaluated the effect of the aptamer on reactive oxygen species (ROS) levels. Intracellular ROS levels reflect the degree of oxidative stress. By measuring ROS levels, the aptamer sequence was further investigated to investigate how it protected cells from MG-induced oxidative stress and enhanced cell viability. As shown in Figure 4J, MG treatment resulted in a 4.6-fold increase in cellular ROS levels, but this trend was significantly alleviated after the addition of the aptamer sequence. These results demonstrate that the MG aptamer has the ability to mask MG toxicity and reduce MG-induced cellular oxidative stress.

[0046] In summary, the superior aptamer sequences demonstrated strong binding to MG in cultured cells, effectively shielding against its toxicity and protecting cells from MG-induced damage. Aptamers reduced the elevated rate of MG-induced apoptosis and mitigated the effects of MG on cell cycle progression, mitochondrial membrane potential, and ROS levels. This protective mechanism may arise from the interaction between the aptamer and MG, thereby influencing MG-mediated cellular redox status and mitochondrial membrane potential. These findings highlight the potential of aptamers in protecting against MG-induced cytotoxicity and demonstrate their potential for application in a variety of biological contexts.

Claims

1. A malachite green nucleic acid aptamer with improved performance, characterized in that: The nucleic acid aptamer sequence is shown in any one of SEQ ID NOs: 2 to 5.

2. The malachite green nucleic acid aptamer according to claim 1, characterized in that The nucleotide sequence of the nucleic acid aptamer is modified and the modified nucleic acid aptamer binds to malachite green, and the modification is selected from at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, and isotopization.

3. A conjugate of malachite green nucleic acid aptamer, characterized in that: The conjugate is a substance for labeling, detection, diagnosis or treatment connected to the nucleotide sequence of the nucleic acid aptamer according to claim 1 or 2, and the conjugate of the nucleic acid aptamer after connection of the substance is combined with malachite green, and the substance is a fluorescent marker such as FAM, a radioactive substance, a therapeutic substance, biotin, digoxin, a nanoluminescent material, a small peptide, siRNA and an enzyme label.

4. Use of the malachite green nucleic acid aptamer according to claim 1 in alleviating the cytotoxicity caused by malachite green, characterized in that: The nucleic acid aptamer is shown in any one of SEQ ID NOs: 2 to 5, and the preferred sequence is SEQ ID NO:

5.

5. The use according to claim 4, characterized in that The alleviating cytotoxicity caused by malachite green refers to the ability to alleviate the effects of malachite green on the cell cycle or cell apoptosis rate or mitochondrial membrane potential or oxidative stress level.

6. The use according to claim 4, characterized in that The malachite green concentration was 1 μM.

7. The use according to claim 4, characterized in that The concentration of the malachite green nucleic acid aptamer is 2-5 μM.

8. A composition for alleviating cytotoxicity caused by malachite green, characterized in that: The composition comprises the malachite green nucleic acid aptamers shown in SEQ ID NOs: 2 to 5 as active ingredients.

9. Use of the composition according to claim 8 in alleviating cytotoxicity caused by malachite green.

10. A preparation for alleviating cytotoxicity caused by malachite green, characterized in that: The preparation contains the malachite green nucleic acid aptamer shown in SEQ ID NO: 2 to 5 as an active ingredient, and the preparation is in the form of tablets, capsules, granules, powders, and pills.

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