Application of malachite green aptamer in low-damage slow-release bacteriostasis and long-acting bacteriostasis

Abstract

The invention discloses an application of a malachite green aptamer in low-damage slow-release bacteriostasis and long-acting bacteriostasis. Specifically, the nucleic acid aptamer with improved affinity, luminescence property and stability can slowly release malachite green in the bacteriostasis process, and assists the long-acting and mild bacteriostasis process. The aptamer with improved multi-dimensional performance is applied to the field of slow-release bacteriostasis, can effectively relieve toxic and side effects of a bactericide on non-target microorganisms or host cells, and is beneficial to realizing long-acting control and relieving drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically the application of malachite green aptamer in low-damage sustained-release and long-lasting antibacterial activity. Background Technology

[0002] Malachite green (MG) is a triphenylmethane compound that was once widely used in aquaculture for antibacterial and antifungal treatment, exhibiting significant bactericidal effects. However, studies have revealed its potential mutagenicity, carcinogenicity, and cumulative toxicity, leading to its ban on use in food-related fields in several countries and regions. Despite this, due to its low cost and ease of use, malachite green continues to be illegally used in some areas, seriously threatening public health and food safety. Therefore, developing highly sensitive, specific, convenient, and efficient detection methods is of great significance for regulating malachite green residues.

[0003] Nucleic acid aptamers, as artificially selected single-stranded DNA or RNA molecules, possess the ability to bind with target molecules with high affinity and high specificity, and are widely used in small molecule detection, bioimaging, and targeted drug delivery. Aptamers targeting malachite green are currently a research hotspot; however, existing aptamers still have shortcomings in affinity, sequence stability, and signal response intensity, limiting their application in the detection of complex samples. Therefore, it is necessary to improve aptamers from multiple dimensions, including structural optimization, screening strategies, and signal coupling mechanisms, to achieve efficient recognition and signal amplification of malachite green, meeting the needs for rapid, accurate, and on-site detection.

[0004] On the other hand, while malachite green exhibits rapid and efficient antibacterial effects in bactericidal applications, its release mechanism is quite aggressive and lacks sustained-release regulation, making it prone to causing toxic side effects on non-target microorganisms or host cells, and hindering long-term control. Furthermore, the unstable release mechanism may exacerbate drug resistance. Therefore, constructing functional modules that can specifically bind to malachite green and regulate its release behavior, such as aptamer-nanocarrier composite systems, can not only help prolong its bactericidal action time and reduce toxicity, but also provide technical support for developing mild and controllable bactericidal systems. Summary of the Invention

[0005] Based on this, the present invention proposes the application of a malachite green aptamer in low-damage sustained-release antibacterial and long-acting antibacterial applications.

[0006] On the one hand, the present invention provides an application of an aptamer in low-damage, long-lasting sustained-release antibacterial agents. The aptamer can specifically bind to the antibacterial agent to form a complex, thereby masking the transient activity of the antibacterial agent and achieving its controlled release. While inhibiting the growth of target microorganisms, it reduces microbial damage or oxidative stress caused by the antibacterial agent, thereby achieving a mild and long-lasting antibacterial effect.

[0007] The antibacterial agent mentioned above is malachite green.

[0008] The aforementioned aptamer forms a complex with malachite green, which reduces the transient antibacterial strength mediated by malachite green and achieves sustained release of malachite green.

[0009] The aforementioned aptamers reduce the level of reactive oxygen species induced by malachite green in microorganisms by binding with malachite green.

[0010] The aforementioned aptamers reduce the malachite green-mediated apoptosis rate of microbial cells by binding to malachite green.

[0011] The aforementioned aptamers maintain the integrity of the target microbial cell morphology during the antibacterial process, reducing cell membrane damage or structural disruption caused by malachite green.

[0012] The aforementioned aptamers reduce the risk of microorganisms developing drug resistance under high antibacterial pressure by prolonging the action time of antibacterial agents.

[0013] The target microorganisms mentioned above are Gram-positive bacteria.

[0014] The aforementioned Gram-positive bacteria is Staphylococcus aureus.

[0015] The aforementioned aptamers are nucleic acid aptamers with high affinity for malachite green, and are any sequence in SEQ ID NO:2 or SEQ ID NO:3.

[0016] On the other hand, the present invention provides an application of malachite green aptamer in mild antibacterial activity, wherein the aptamer is any sequence in SEQ ID NO:2 or SEQ ID NO:3.

[0017] The present invention also provides an application of malachite green aptamer in extending the shelf life of antibacterial agents, wherein the aptamer is any sequence in SEQ ID NO:2 or SEQ ID NO:3.

[0018] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention obtains malachite green DNA aptamers with comprehensively improved fluorescence performance, stability, affinity, and binding ability through multi-dimensional systematic tailoring; 2. This invention applies multi-dimensional performance-enhancing aptamers to the field of mild antibacterial agents, which can effectively alleviate the toxic side effects of bactericides on non-target microorganisms or host cells, and facilitate long-term control and reduce drug resistance.

[0019] 3. This invention utilizes the high affinity and specific binding between the multidimensional performance-enhanced aptamer and the antibacterial agent to form a stable aptamer-antibacterial agent complex, thereby effectively masking the transient activity of the antibacterial agent and achieving controlled sustained release and long-term release of the antibacterial agent. While ensuring the antibacterial effect, it significantly reduces the level of antibacterial agent-induced microbial oxidative stress, cell damage and apoptosis, achieving a mild and low-damage antibacterial mode.

[0020] 4. The aptamer sustained-release antibacterial strategy proposed in this invention can maintain a stable antibacterial effect over a longer timescale, avoiding the instantaneous high antibacterial pressure brought about by traditional high-dose, strong bactericidal methods. This helps to maintain the balance of the microbial ecosystem and reduce the risk of drug-resistant strains, expanding the application potential of antibacterial agents in the fields of chronic infection management, medical material surface protection, and biosafety. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of stem trimming. A shows the secondary structures of different MG DNA aptamer variants during stem optimization, illustrating the minimum free energy and base pairing probability. Purple nucleotides indicate the optimized positions. B is a circular dichroism chromatogram of the optimized stem trimming sequence. C shows the fluorescence activation effect of different MG DNA aptamer variants after stem trimming. The dashed line represents the fluorescence intensity of the original sequence (set as 1× fluorescence). D–G represent the three-dimensional structures of the complexes of CAG-T-ML-S4, CAG-T-ML-S4-TA, CAG-T-ML-S4-TA-AT, and CAG-T-ML-S4-TA-AT+1 with MG, respectively, and the view after rotating 90° (top). The enlarged box in the middle highlights the MG binding pocket. The bottom is a two-dimensional schematic diagram of molecular docking at the MG binding site.

[0022] Figure 2 The secondary structures of different MG DNA aptamer variants during the top stem optimization process are shown, along with the minimum free energy and base pairing probability for each structure. Purple bases indicate the optimized positions.

[0023] Figure 3 The secondary structures of different MG DNA aptamer variants during the bottom stem sequence optimization process are shown, along with the minimum free energy and base pairing probability for each structure. Purple bases indicate the optimized positions.

[0024] Figure 4The secondary structures of different MG DNA aptamer variants during the bottom stem length optimization process are shown, along with the minimum free energy and base pairing probability for each structure. Purple bases indicate the optimized positions.

[0025] Figure 5 This diagram illustrates loop tandem trimming. A shows the fluorescence activation effect of different MG DNA aptamer variants during loop tandem optimization. The dashed line represents the fluorescence intensity of the original sequence (set as 1× fluorescence). The inset shows the agarose gel electrophoresis results corresponding to these sequences; B shows the secondary structures of different MG DNA aptamer variants after loop tandem optimization, with minimum free energy and base pairing probabilities. Purple nucleotides indicate the optimized positions; C shows the first ten molecular docking models of the three-dimensional structures of the CAG-T-ML-S4-TA-AT+1-1L to 6L sequences with the MG complex.

[0026] Figure 6 The results show the molecular docking of the CAG-T-ML-S4-TA-AT+1-1L sequence with MG. A shows the three-dimensional structure of the sequence-MG complex and its rotation by 90°; B shows a two-dimensional schematic diagram of the molecular docking of the MG molecule at the sequence binding site; C shows a three-dimensional schematic diagram of the MG binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0027] Figure 7 The results show the molecular docking of the CAG-T-ML-S4-TA-AT+1-3L sequence with MG. A shows the three-dimensional structure of the sequence-MG complex and its rotation by 90°; B shows a two-dimensional schematic diagram of the molecular docking of the MG molecule at the sequence binding site; C shows a three-dimensional schematic diagram of the MG binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0028] Figure 8 The results show the molecular docking of the CAG-T-ML-S4-TA-AT+1-4L sequence with MG. A shows the three-dimensional structure of the sequence-MG complex and its rotation by 90°; B shows a two-dimensional schematic diagram of the molecular docking of the MG molecule at the sequence binding site; C shows a three-dimensional schematic diagram of the MG binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0029] Figure 9 The results show the molecular docking of the CAG-T-ML-S4-TA-AT+1-5L sequence with MG. A shows the three-dimensional structure of the sequence-MG complex and its rotation by 90°; B shows a two-dimensional schematic diagram of the molecular docking of the MG molecule at the sequence binding site; C shows a three-dimensional schematic diagram of the MG binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0030] Figure 10The results show the molecular docking of the CAG-T-ML-S4-TA-AT+1-6L sequence with MG. A shows the three-dimensional structure of the sequence-MG complex and its rotation by 90°; B shows a two-dimensional schematic diagram of the molecular docking of the MG molecule at the sequence binding site; C shows a three-dimensional schematic diagram of the MG binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0031] Figure 11 The structural properties of the ring-tandem sequence were evaluated. A–F represent Job's curves for the CAG-T-ML-S4-TA-AT+1-1L–6L sequences, respectively. The dashed line represents the intersection of the two fitted lines, and the x-axis values ​​of the intersection points are marked in the figure. G–K represent the three-dimensional structures (left) of the MG binding pockets in the CAG-T-ML-S4-TA-AT+1-1L, -3L, -4L, -5L, and -6L sequences, respectively, and the two-dimensional molecular docking schematic diagrams of the corresponding MG binding sites (right). L–Q represent the circular dichroism chromatograms of the CAG-T-ML-S4-TA-AT+1-1L–6L sequences, respectively.

[0032] Figure 12 To verify aptamer affinity using the MST method, sequence A is CAG-T-ML-S4-TA-AT+1; sequence B is CAG-T-ML-S4-TA-AT+1-4L.

[0033] Figure 13 To verify the affinity of the aptamer using the colloidal gold method. A is the CAG-T-ML-S4-TA-AT+1 sequence; B is the CAG-T-ML-S4-TA-AT+1-4L sequence.

[0034] Figure 14 The results are as follows: A is the circular dichroism chromatogram of the CAG-T-ML-S4-TA-AT+1-4L sequence after interaction with MG or ThT; B is the fluorescence detection result of cation optimization in the biosensor; C is the circular dichroism chromatogram of the CAG-T-ML-S4-TA-AT+1-4L sequence under different cation conditions; D is the fluorescence detection result of pH optimization in the biosensor; E is the detection waveform of the CAG-T-ML-S4-TA-AT+1-4L sequence biosensor; F is the linear relationship of the CAG-T-ML-S4-TA-AT+1 and CAG-T-ML-S4-TA-AT+1-4L sequence biosensors.

[0035] Figure 15 This demonstrates the detection specificity of the MG proportional sensor.

[0036] Figure 16 The detection waveform of the CAG-T-ML-S4-TA-AT+1 sequence biosensor is shown.

[0037] Figure 17 This study evaluates the sustained-release and long-lasting bactericidal effects of nucleic acid aptamers on MG at the microbial level. A shows an image of the inhibition zone of Staphylococcus aureus on a culture plate after treatment with MG and different nucleic acid sequences; B shows the quantitative analysis of the diameter of the inhibition zone; C shows the quantitative results of reactive oxygen species (ROS) fluorescence levels under different treatment conditions; D shows the quantitative results of the apoptosis rate of microorganisms under different treatment conditions; E-H show scanning electron microscopy (SEM) images of Staphylococcus aureus under different treatment conditions. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1. Trimming of the aptamer stem The nucleotide sequences involved in the experiment are shown in Table 1.

[0040] Table 1. Nucleotide sequences involved in the experiment

[0041] Using the MG DNA aptamer as the initial aptamer (Original sequence), the stem sequence was optimized by substitution. The original stem bases of the CAG-T-ML-S4 sequence were AT complementary base pairs, which were replaced with TA, GC, CG, AT interpolation, and CG interpolation. Figure 1 A, Figure 2 The results showed that replacing all the top stem base pairs with TA significantly enhanced the fluorescence excitation of MG. Figure 1 C). Furthermore, the fluorescence excitation of MG by the sequence was significantly reduced after the mutation to GC base pairs. This indicates that the apical stem not only provides secondary structural support, but its sequence itself participates in the formation of the MG-specific binding pocket. In addition, molecular docking results showed that replacing the apical stem with TA base pairs caused the MG binding pocket to shift towards the apex, and the TA stem participated in the MG interaction (…). Figure 1 E). This indicates that the CAG-T-ML-S4-TA sequence can interact with MG in a more affinity and specific manner, and its higher-order structure stability is also superior to that of CAG-T-ML-S4 (E). Figure 1 A).

[0042] The basal stem sequence of the aptamer was further optimized. Five aptamer variants exist: the original basal stem (CAG-T-ML-S4-TA), AT, TA, GC, and CG basal stem (…). Figure 1 A, Figure 3 The results showed that the CAG-T-ML-S4-TA-AT sequence had the best fluorescence excitation effect on MG, but other sequences also had relatively superior fluorescence excitation ability for MG. Figure 1 C). It's possible that because the basal stem is far from the binding pocket of MG, the type of basal stem sequence does not significantly affect MG binding. The basal stem likely only provides a stable aptamer conformation and topology. Molecular docking results show that the AT base pair at the bottom of the CAG-T-ML-S4-TA-AT sequence increases the number of interacting bases with MG, and the interacting bases and interaction pockets are mainly concentrated in the top loop. Specifically, A24 interacts with MG via hydrophobicity, G7 and T9 via hydrogen bonds, and T8, T10, A21~A23, and A25 via van der Waals forces. The basal stem, however, does not interact with the MG molecule, which explains why the type of basal stem sequence has little effect on MG fluorescence excitation. Figure 1 F).

[0043] The experiment further optimized the number of base pairs in the basal stem by adding 1-5 pairs or deleting 1-5 pairs of complementary AT base pairs to the CAG-T-ML-S4-TA-AT sequence, resulting in the CAG-T-ML-S4-TA-AT+1-5 and CAG-T-ML-S4-TA-AT-1-5 sequences. Figure 1 A, Figure 4 ).like Figure 1 As shown in Figure C, fluorescence results indicate that the fewer complementary base pairs in the bottom stem, the weaker the fluorescence, suggesting that its secondary structure may have been disrupted, preventing MG from embedding into the fluorescent pocket and thus hindering fluorescence excitation. Furthermore, the CAG-T-ML-S4-TA-AT+1 sequence showed the best fluorescence excitation effect for MG; increasing the number of AT base pairs by 2-5 pairs resulted in a decreasing trend in fluorescence. This may be due to excessively long complementary base pairs, leading to overly stable bottom stem pairing, reduced sequence flexibility, and a lack of conformational folding space, preventing the higher-order structure of the sequence from forming space for MG embedding. Molecular docking also indicated that the TA-AT+1 sequence retained the binding pocket and recognition function of the TA stem at the top of the middle loop. Figure 1 G).

[0044] In terms of circular dichroism (CD) spectroscopy, the CD spectrum of the purine-pyrimidine complex poly[d(A)]·poly[d(T)] differs from that of the B-DNA helical structure. The former exhibits an unusual B'-DNA structure, showing a positive peak around 280 nm, a negative peak around 245 nm, and a positive peak around 220 nm. For example... Figure 1 As shown in Figure B, with the increase of AT and TA base pairs in the sequence, the positive peak at 220 nm in the CD spectrum of the sequence becomes higher and higher. With the increase of (A+T) content, the negative band of the CD spectrum of the sequence becomes deeper, and the conformational variability increases.

[0045] Furthermore, the hairpin structure is an intramolecular variant of B-DNA. In addition to the double helix stem, it also contains a single-stranded loop, thus exhibiting a shoulder peak at approximately 260 nm. Compared to the CAG-T-ML-S4 sequence, the other three optimized sequences obtained through stem optimization all showed varying degrees of shoulder peaks at 260 nm, indicating a change in the higher-order topology of the optimized sequences. Moreover, CD spectroscopy produced significant hairpin structure features, suggesting different hairpin structure formation efficiencies among different sequences. Figure 1 B).

[0046] Example 2. Optimized trimming of the ring section of the aptamer The successful replication of the TGT-AAA loop suggests that experiments should further explore tandem loops. During the trimming process, the top loop was replaced with the original ligand loop, yielding a single-loop sequence, CAG-T-ML-S4-TA-AT+1-1L. The CAG-T-ML-S4-TA-AT+1 sequence is equivalent to CAG-T-ML-S4-TA-AT+1-2L. Further tandem multivalent TGT-AAA loops yielded trivalent, tetravalent, pentavalent, and hexavalent sequences, namely the CAG-T-ML-S4-TA-AT+3L~6L sequences. Figure 5 B). For example... Figure 5 As shown in A and B, the fluorescence results indicate that the sequence stability increases with the increase of the number of tandem TGT-AAA rings, and the fluorescence excitation of the sequence pair with MG gradually strengthens. When tandemly connected to four rings, the fluorescence of the CAG-T-ML-S4-TA-AT+1-4L sequence essentially reaches a plateau. This demonstrates that the TGT-AAA ring formed based on GA mismatch and TA complementarity can indeed specifically enhance MG excitation, and this effect can be superimposed through the tandem connection of functional units. Of course, the TA complementarity stem around the TGT-AAA ring cannot be ignored; besides stabilizing the secondary structure of the aptamer, it also forms the binding pocket of MG, providing dye insertion sites. Furthermore, as... Figure 1 G, Figure 5 C Figures 6-10 and Figure 11 The molecular docking results shown in G-K indicate that MG preferentially binds to the top of the rings closest to the 5' and 3' ends. Furthermore, the TA stem between each pair of rings is actually a potential MG binding pocket. The tandem TGT-AAA rings provide a richer array of binding sites for MG, potentially altering the stoichiometry of the aptamer-target interaction and thus leading to increasingly higher fluorescence quantum yields.

[0047] The results of circular dichroism chromatogram show that ( Figure 11 (L~Q), as the number of tandem loops increases, the sequence still maintains positive peaks at 220 and 280 nm and a negative peak at 245 nm, and the shoulder peak at 260 nm becomes increasingly prominent. These results indicate that the tandem loop sequence exhibits a typical mixed structure of hairpins and B-DNA helices.

[0048] Example 3. Identification of the binding performance of aptamers 1. Chemistry stoichiometry evaluation Table 2 Stoichiometry of MG DNA aptamer variants binding to MG

[0049] The experiment further tested the binding molar ratios of MG to the six sequences using the Job's plot method. The results showed that the fluorescence peaks in the fitted plots of the six sequences CAG-T-ML-S4-TA-AT+1-1L to 6L appeared at 0.315, 0.257, 0.215, 0.208, 0.205, and 0.117, respectively. Figure 11 (A-F). As the number of tandem intermediate rings increases, the X value corresponding to the intersection of the two fitted lines gradually decreases, exhibiting an increasingly higher stoichiometry (Table 2). From the CAG-T-ML-S4-TA-AT+1-1L sequence to the -5L sequence, one sequence can bind to approximately 2-4 MG molecules, while a hexacyclic sequence can bind to 7.636 MG molecules. The significantly improved stoichiometry also explains why the more tandem rings there are, the stronger the fluorescence.

[0050] 2. Affinity Assessment Aptamer affinity is one of its most important properties. We then used various methods to monitor the affinity of the CAG-T-ML-S4-TA-AT+1 and CAG-T-ML-S4-TA-AT+1-4L sequences. According to the literature, the affinity Kd value of the original sequence is 2.43 µM. For example... Figure 12 As shown, the sequence affinity of CAG-T-ML-S4-TA-AT+1 and CAG-T-ML-S4-TA-AT+1-4L gradually increases, while the affinity gradually decreases.K d Value. The colloidal gold results also showed the same trend ( Figure 13 Both affinity determination methods demonstrated that the cut sequence had a significantly higher affinity than the original sequence.

[0051] Example 4. Multidimensional performance enhancement aptamers for malachite green biosensors The potential of the pruned sequence in detection was further explored. A label-free proportional aptamer sensor was constructed using the competitive equilibrium principle. The aptamer binds to MG molecules and induces MG fluorescence emission. A competitive fluorescent molecule, ThT, is added to the aptamer-MG complex system. ThT competes with MG for the same nucleic acid binding sites, leading to a change in the fluorescence intensity of the system, thus enabling the sensing and detection of malachite green.

[0052] ThT, as a dye molecule, can embed into higher conformations such as G tetrands, double strands, and hairpin structures, thereby achieving enhanced fluorescence excitation. In the MG sensor, a proportional, label-free, ultrasensitive, and ultrafast biosensor was constructed using MG and ThT dyes. Because the CAG-T-ML-S4-TA-AT+1-4L sequence exhibits relatively high fluorescence excitation, it was chosen for further validation and condition optimization. Circular dichroism spectroscopy results showed that the CD spectra of the CAG-T-ML-S4-TA-AT+1-4L sequence shifted to varying degrees after the addition of MG and ThT, respectively, and exhibited distinct shoulder peaks, indicating that both small molecules can bind and interact with the aptamer. Figure 14 A).

[0053] Furthermore, to obtain optimal sensing performance, the experimental conditions were systematically optimized. The sensor exhibited fluorescence signals of varying intensities under different ion conditions. Figure 14 B). This is because ionic conditions can significantly affect the conformation of nucleic acids. CD spectroscopy shows that when no ions are added or when potassium, sodium, and magnesium ions are present in the system, nucleic acids can maintain their higher-order structure without being destroyed. However, when lead ions are present in the system, the higher-order conformation of the nucleic acid completely collapses, which also explains the sharp drop in fluorescence intensity of the dye molecules in this system. Figure 14 (B and C). However, the ratio of MG and ThT fluorescence peaks did not change significantly under different ion conditions, highlighting the unique advantage of the proportional sensor and demonstrating its resistance to ion interference. Considering both fluorescence intensity and the ratio of the fluorescence peaks of the two dye molecules, the optimal detection condition was chosen to avoid introducing ions. Furthermore, the proportional sensor also exhibits excellent resistance to pH interference, maintaining a relatively stable signal value within the pH range of 5.4–8.4. Finally, a mild condition of pH 7.4 was selected as the pH for the sensing process. Figure 14F). Evaluation showed that the proportional sensor constructed using CAG-T-ML-S4-TA-AT+1-4L exhibited good linearity over a wide range of 5 nM to 8 µM, achieving a low detection limit of 1.12 nM (F). Figure 14 E and F). Furthermore, the constructed proportional sensor exhibits superior specificity for crystal violet (CV), rhodamine B (RB), Coomassie Brilliant Blue (CBB), and colorless malachite green (LMG) fuels. Figure 15 The proportional sensor constructed using the CAG-T-ML-S4-TA-AT+1 sequence also demonstrated good MG sensing potential, and the detection performance of different sequences could meet application requirements. Figure 14 F, Figure 16 (Table 3).

[0054] Table 3. Biosensor performance of different MG sequences

[0055] In spiked recovery experiments on real lake water and fish meat samples, the proposed proportional biosensor showed good recovery rates, demonstrating its applicability in real sample analysis and its adaptability to common sample matrices and potential interferences (Table 4). These results demonstrate the application potential of different aptamer variants and the versatility of the applied biosensing strategy.

[0056] Table 4 Results of Real Sample Recovery Tests

[0057] Example 5. Application of aptamers with multidimensional performance enhancement in mild antibacterial activity. "Mild antibacterial action" is a type of antibacterial strategy that differs from traditional strong bactericidal strategies. It emphasizes minimizing damage to the host environment, micro-ecosystem, or non-target microorganisms while inhibiting the excessive growth or infection of microorganisms. This strategy typically does not aim to completely kill the target microorganism in a short period of time, but rather achieves a long-lasting, sustained-release, minimally damaging, and low-inducible antibacterial mechanism by controlling the release of antibacterial factors, reducing the instantaneous peak concentration of antibacterial agents, or masking their activity.

[0058] Mild antimicrobial agents are of great significance. They slow the development of drug resistance: Strong and high-dose bactericidal pressure is one of the main causes of bacterial resistance. Mild antimicrobial agents significantly reduce the risk of stress-induced mutations in microorganisms by reducing transient drug stress, thus reducing the emergence of resistant strains at the source. They maintain microecological balance: Mild antimicrobial agents, while not completely eliminating the target microbiota, preserve the activity of beneficial or neutral microorganisms in the environment, helping to maintain the functional integrity of complex micro-ecosystems such as the gut and skin. They reduce host toxicity and side effects: High concentrations of antimicrobial agents may cause side effects such as oxidative stress and membrane damage to host cells (e.g., skin cells, intestinal epithelial cells), while mild antimicrobial agents help reduce non-targeted damage to host cells, making them particularly suitable for long-term exposure or chronic disease states. They are more suitable for the management of chronic, low-grade infections: For certain non-acute infection states (e.g., wound care, skin inflammation, oral plaque control, medical device surface management), mild antimicrobial agents can provide a physiologically friendly management approach.

[0059] The "mild antibacterial" effect has numerous potential applications. Medical dressings or implant materials: By using aptamers to control the release of antibacterial agents, infections can be prevented in surgical areas or on implant surfaces, while avoiding cytotoxicity or tissue irritation. Daily care products: In areas such as skincare and oral care, mild antibacterial strategies can be used to control bacterial growth without disrupting the normal microbial barrier. Chronic wound management: Particularly suitable for chronic wounds such as diabetic foot ulcers and pressure ulcers, achieving antibacterial effects while avoiding irritation or damage to newly formed tissues. Bioregulation in microbial culture / synthetic biology platforms: Controllable inhibition of contaminating bacteria or regulation of population density can be used for high-precision synthetic biology regulation and microbial community design. Agriculture and food safety: Can be used on fruit and vegetable surfaces, packaging materials, or hydroponic systems to provide low-residue, environmentally friendly antibacterial protection.

[0060] Against this backdrop, the experiment further extended the potential of aptamers with multidimensional performance enhancements to the field of microbial applications, attempting to utilize the binding ability of aptamers to MG to achieve sustained-release antibacterial effects. MC-DA, MC-DA-LG, CAG-T-ML-S4, and CAG-T-ML-S4-TA-AT+1-4L were selected as intervention sequences.

[0061] MG is a potent antibacterial agent that significantly inhibits both Gram-positive and Gram-negative bacteria. Figure 17As shown in Figure A, adding MG solution to the small well in the center of a culture plate inoculated with Staphylococcus aureus produced a significant inhibition zone on the first day, indicating that MG can indeed significantly inhibit the growth of Staphylococcus aureus. The addition of the aptamer sequence resulted in a significantly smaller inhibition zone compared to the MG-only group. A smaller inhibition zone diameter indicates a certain degree of reduced bactericidal effect. Furthermore, on the first day, as the affinity between the aptamer sequence and MG increased, the diameter of the inhibition zone gradually decreased. Figure 17 (A and B). This indicates that a tighter binding of the aptamer to MG can enhance the masking of the bactericidal effect of MG. In all groups, the inhibition zone gradually decreased from day 1 to day 5, but the decrease in the inhibition zone on day 5 was less pronounced than that on day 4. On day 5, the inhibition zone in the aptamer group was smaller than that in the MG group, indicating that the aptamer can protect microorganisms from the potent damage of the bactericide over a period of several days, thus providing a slow-release and long-lasting bactericidal process.

[0062] It is evident that the aptamer forms a complex upon binding with MG, which can mask the damaging effects of MG on microorganisms. As the aptamer gradually degrades in the system, more and more naked MG molecules are exposed, thus achieving a slow-release and long-lasting bactericidal effect. This effect can be applied to various scenarios; that is, using the same amount of bactericide may help achieve a milder and longer-lasting bactericidal effect.

[0063] Furthermore, the effects of MG aptamer sequences on microbial redox stress and survival were investigated. Figure 17 As shown in Figure C, the addition of the antibacterial agent MG significantly increased the ROS level of the microorganisms, which was 2.68 times that of the control group. The addition of different aptamer sequences significantly reduced the ROS level of the microorganisms. With further aptamer optimization, the sequence affinity became stronger, and the ROS level decreased. The ROS level of the microorganisms in the MG@CAG-T-ML-S4-TA-AT+1-4L sequence group was not significantly different from that of the control group, indicating that the stronger the aptamer affinity, the more effectively it can alleviate and reduce the MG-mediated oxidative stress effect.

[0064] A similar trend was observed when assessing the apoptosis rate of Staphylococcus aureus. The addition of the aptamer sequence could alleviate MG-mediated microbial apoptosis in the short term; however, the apoptosis rates in the MG@ML-S4 and MG@CAG-T-ML-S4-TA-AT+1-4L sequence treatment groups were not significantly different from the control group. Figure 17 D). This further illustrates the role of aptamer sequences in the sustained release and long-lasting bactericidal effect of antibacterial agents.

[0065] To systematically evaluate the effect of aptamers on the bactericidal effect mediated by antibacterial agents, we further observed the morphology of Staphylococcus aureus in different treatment groups using SEM at the morphological level. For example... Figure 17 As shown in Figure E, the Staphylococcus aureus in the control group exhibited a regular spherical shape, uniform individual morphology, smooth surface, clear outline, and intact structure, without damage, collapse, or abnormal protrusions, indicating that the microbial structure maintained good integrity and presented healthy morphological characteristics. After the addition of MG, the morphology of the cocci changed significantly, no longer exhibiting a regular spherical shape; some cells collapsed, deformed, and even formed irregular cystic structures. There was significant morphological variation between individuals, disrupting the homogeneity of the population. Furthermore, the surface of the cocci became rough, possibly indicating damage to the membrane structure, leading to an inability to maintain normal cell morphology. Their basic structure was completely destroyed, presenting an unhealthy physiological state. Figure 17 F and G). After the addition of the aptamer, the perforation morphology of the microorganisms was greatly alleviated, exhibiting a morphological structure similar to that of the control group. Figure 17 H). This experiment demonstrates from a morphological perspective that aptamers provide some protection against microorganisms treated with antibacterial agents.

[0066] In summary, this invention tested the multidimensionally enhanced aptamer sequence on microbial cultures and found that it can effectively bind to MG molecules, thereby releasing MG in a controlled and gradual manner over a period of time. This sustained-release mechanism enables the aptamer to exert a long-lasting antibacterial effect, providing a new method for mitigating microbial infections while minimizing the risk of drug resistance.

Claims

1. The use of aptamer in the low-damage long-acting sustained-release bacteriostasis of bacteriostatic agents, characterized in that, The aptamer can specifically bind with the bacteriostatic agent to form a complex, mask the transient activity of the bacteriostatic agent, and achieve controllable release of the bacteriostatic agent, thereby achieving a mild and long-acting bacteriostatic effect by inhibiting the growth of target microorganisms while reducing the microbial damage or oxidative stress caused by the bacteriostatic agent.

2. Use according to claim 1, characterized in that, The bacteriostatic agent is malachite green.

3. Use according to claim 1 or 2, characterized in that, The aptamer binds with the malachite green to form a complex, reduces the transient bacteriostatic intensity mediated by the malachite green, and achieves sustained and slow release of the malachite green.

4. The use according to any one of claims 1 to 3, characterized in that, The aptamer binds with the malachite green to reduce the level of reactive oxygen species of microorganisms induced by the malachite green.

5. The use according to any one of claims 1 to 4, characterized in that, The aptamer binds with the malachite green to reduce the apoptosis rate of microorganisms mediated by the malachite green.

6. The use according to any one of claims 1 to 5, characterized in that, The aptamer maintains the integrity of the cell morphology of the target microorganism during the bacteriostatic process, and reduces the cell membrane damage or structural damage caused by the malachite green.

7. The use according to any one of claims 1 to 6, characterized in that, The aptamer prolongs the action time of the bacteriostatic agent, and reduces the risk of drug resistance of microorganisms under high bacteriostatic pressure.

8. The use according to any one of claims 1 to 7, characterized in that, The target microorganism is a gram-positive bacterium.

9. Use according to claim 8, characterized in that, The gram-positive bacterium is Staphylococcus aureus.

10. Use according to any one of claims 1 to 9, characterized in that, The aptamer is a nucleic acid aptamer with high affinity binding capacity for malachite green, and is any one of SEQ ID NO: 2 or SEQ ID NO: 3.

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