An antisense oligonucleotide sequence targeting the ftsZ gene of Gram-positive bacteria

Abstract

The present invention discloses an antisense oligonucleotide targeting the ftsZ gene of Gram-positive bacteria, wherein the antisense oligonucleotide sequence is: 5'-TTGTCAACGA-3'; the antisense oligonucleotide sequence of the present invention takes the ftsZ gene of Staphylococcus aureus and its drug-resistant bacteria as the target, interferes with the expression of genes essential for the survival of Staphylococcus aureus and its drug-resistant bacteria through the Watson-Crick base complementary pairing principle, thereby exerting an antibacterial effect.

Description

Technical Field

[0001] The invention belongs to the technical field of therapeutic activity of biomedical compounds and discloses an antisense oligonucleotide sequence targeting the ftsZ gene of Gram-positive bacteria. Background Art

[0002] Staphylococcus aureus (S. aureus) is the leading cause of community-associated infections and can lead to many fatal infectious diseases such as endocarditis, pneumonia, bacterial anemia, etc. Worse, it is reported that methicillin-resistant Staphylococcus aureus (MRSA) accounts for about 25%-50% of nosocomial S. aureus infections, causing significant morbidity, mortality and huge economic burden.

[0003] Existing antibacterial drugs are all traditional small molecule antibacterial drugs. In recent years, the rapid discovery and reporting of drug-resistant bacteria caused by the abuse of antibiotics, as well as the slow development of new antibiotics, have made it difficult for humans to treat bacterial infections, and the drug resistance situation has become increasingly severe. Therefore, there is an urgent need to develop innovative antibacterial strategies to treat MRSA infections. Summary of the invention

[0004] The purpose of the present invention is to provide an antisense oligonucleotide sequence targeting the ftsZ gene of Gram-positive bacteria, which inhibits the growth of bacteria by inhibiting the expression of the target gene and has a good inhibitory effect on infection by Staphylococcus aureus and drug-resistant bacteria thereof.

[0005] The present invention adopts the following technical scheme: an antisense oligonucleotide sequence targeting the ftsZ gene of Gram-positive bacteria, the antisense oligonucleotide sequence is: 5'-TTGTCAACGA-3'.

[0006] Another technical solution of the present invention: use of the above antisense oligonucleotide sequence in the preparation of drugs for inhibiting Staphylococcus aureus and its drug-resistant bacteria.

[0007] Furthermore, the above antisense oligonucleotide sequence is used to inhibit the expression of ftsZ gene of Staphylococcus aureus and its drug-resistant bacteria.

[0008] Another technical solution of the present invention: a drug for inhibiting Staphylococcus aureus and its drug-resistant bacteria, comprising the above-mentioned antisense oligonucleotide sequence.

[0009] The beneficial effect of the present invention is that the antisense oligonucleotide sequence of the present invention targets the ftsZ gene of Staphylococcus aureus and its drug-resistant bacteria, interferes with the expression of genes essential for the survival of Staphylococcus aureus and its drug-resistant bacteria through the Watson-Crick base complementary pairing principle, thereby exerting an antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of the ftsZ antisense region of the antisense oligonucleotide sequence in Staphylococcus aureus (ATCC29213) and its drug-resistant bacteria (USA300 LAC) in the embodiments of the present invention;

[0011] Figure 2 Schematic diagram of the secondary structure of ftsZ gene mRNA in an embodiment of the present invention;

[0012] Figure 3 Schematic diagram of growth curves of different sequences in the embodiments of the present invention;

[0013] Figure 4 Schematic diagram of the particle size of DP-AD7 nanoparticles in an embodiment of the present invention;

[0014] Figure 5 Schematic diagram of the morphology of DP-AD7 nanoparticles in an embodiment of the present invention;

[0015] Figure 6 This is a schematic diagram of the DP-AD7 bacterial delivery efficiency in an embodiment of the present invention;

[0016] Figure 7 This is a schematic diagram of real-time observation of the efficiency of bacteria taking up nanoparticles in an embodiment of the present invention;

[0017] Figure 8 This is a schematic diagram of the growth inhibition of S. aureus (ATCC29213) in the embodiments of the present invention;

[0018] Fig. 9 This is a schematic diagram of the growth inhibition of MRSA (USA300 LAC) in an embodiment of the present invention;

[0019] Fig.10 Schematic diagram of the inhibition of S. aureus (ATCC29213) ftsZ gene expression in the embodiment of the present invention;

[0020] Fig.11 Schematic diagram of the inhibition of MRSA (USA300 LAC) ftsZ gene expression in an embodiment of the present invention. DETAILED DESCRIPTION

[0021] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Traditional small molecule antibacterial drugs have a long development cycle, are prone to drug resistance, have many adverse reactions, and the development speed is far behind the speed of drug-resistant bacteria. Antisense antibacterial agents are macromolecular biological drugs composed of at least 8 antisense oligonucleotides (ASOs), which bind to target RNA and regulate the function of RNA through several different mechanisms. ASOs interfere with the expression of target genes such as essential genes for bacterial survival, virulence or resistance-related genes through the Watson-Crick base complementary pairing principle, and exert antibacterial effects. Compared with traditional antibacterial drugs, their targets are clear, and their development model is relatively fixed, which can quickly respond to emerging drug-resistant bacteria. The filamentous GTPase encoded by ftsZ is an essential protein for the survival of Staphylococcus aureus, and it is well conserved and has high homology in Gram-positive bacteria such as Staphylococcus aureus, indicating that this gene is a suitable target for ASOs. Therefore, antisense antibacterial drugs targeting ftsZ have become an ideal choice for combating drug-resistant bacterial infections.

[0023] The invention discloses an antisense oligonucleotide sequence (Antisense Oligonucleotides, ASOs) targeting gram-positive bacteria ftsZ gene, and the antisense oligonucleotide sequence is: 5'-TTGTCAACGA-3'.

[0024] The antisense oligonucleotide sequence of the present invention targets the ftsZ gene of Staphylococcus aureus and interferes with the expression of genes essential for the survival of Staphylococcus aureus through the Watson-Crick base complementary pairing principle, thereby exerting an antibacterial effect and treating infections caused by Staphylococcus aureus and its drug-resistant bacteria.

[0025] In one embodiment, Figure 1 The figure shows the schematic diagram of the ftsZ antisense region of Staphylococcus aureus (ATCC29213) and its resistant bacteria (USA300LAC). It can be seen from the figure that the sequence of the ftsZ antisense region (gray part) of Staphylococcus aureus (ATCC29213) and its resistant bacteria (USA300 LAC) is the same.

[0026] The homology comparison analysis between Staphylococcus aureus (ATCC29213, CP094857.1) and other Staphylococci aureus is shown in Table 1. ftsZ is well conserved in Staphylococcus aureus and its resistant bacteria.

[0027] Table 1

[0028]

[0029] In one embodiment, the design and screening methods of the antisense oligonucleotide sequences are disclosed.

[0030] 1. Reagents and strains.

[0031] 1.1 MRSA (USA300 LAC) purchased from the National Institutes of Health in the United States and S. aureus (ATCC29213) (sensitive strain) preserved in the laboratory were used as strains in this experiment.

[0032] Recovery: Take the experimental strain out of the -80℃ refrigerator, streak it on the MH agar plate, culture it in a 37℃ bacterial incubator for 24 hours, randomly pick a single clone on the agar plate and add it to 3mL TSB broth, culture the bacteria at 37℃ and 270rpm for about 8-10 hours until the logarithmic growth phase, and set aside. Adjust the bacterial solution concentration: Add 150μL of MH broth to each well of the 96-well plate in turn, take 150μL of bacterial solution and dilute it in multiples and discard it, and finally add MH broth to each well to 300μL, use a multi-function microplate reader to measure the OD value at 630nm, and the bacterial solution concentration is about 10 when the OD value is 0.1. 8 CFU / mL.

[0033] 1.2 MH broth medium: Weigh about 2.5 g of MH broth medium powder, add 100 mL dd H2O until completely dissolved, place in a high pressure steam sterilizer, autoclave at 121℃ for 20 min, cool and store in a 4℃ refrigerator for later use.

[0034] 1.3 Tryptone soy broth (TSB): Weigh about 2.5 g of TSB dry powder, add 250 mL of dd H2O, sterilize using the same method, cool and store in a refrigerator at 4°C for later use.

[0035] 1.4 ASOs were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The ASOs in the embodiments of the present invention refer to the above-mentioned antisense oligonucleotide sequence, i.e., 5'-TTGTCAACGA-3'. DPP7 (dendritic poly-peptides 7) was commissioned to be synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., where DPP7 is a branched polypeptide, which was published in "Amphipathic dendritic poly-peptides carrier to deliver antisense oligonucleotides against multi-drugresistant bacteria in vitro and in vivo" in Journal of Nanobiotechnology (2022).

[0036] 1.5 Preparation of DPP7, ASOs, and DSPE-mPEG2000 working solutions:

[0037] Take 2mg of DPP7, dissolve and mix with 33.3μL and 29.4μL of 25% acetonitrile respectively, and obtain a mother solution of 20mM, which is stored at -80℃ for use. Dilute the DPPs mother solution 25 times with sterile water to obtain a working solution of 0.8mM, 100μL / tube, and store at -80℃ for use. Take 10.1nmol of ASOs and add 505μL dd H2O to obtain a 20μM ASOs working solution for use. Weigh 2.8mg DSPE-mPEG2000, dissolve in 1mL dd H2O to obtain a 100mM storage solution, then take 20μL and add it to 980μL dd H2O to obtain a working solution with a concentration of 20μM, and store it at -20℃ for use.

[0038] 1.6 Preparation of DP-AD7 (i.e., DPP / ASOs nanoparticles modified with DSPE-mPEG2000):

[0039] Take 7.5 μL DPP7 working solution, 50 μL ASOs and 917.5 μL dd H2O and incubate at 37°C for 30 minutes, then add 25 μL DSPE-mPEG2000 and incubate at 37°C for 30 minutes to obtain 1 μM DP-AD. DP-AD7 nanoparticles of other concentrations were prepared according to the same process and ratio.

[0040] 1.7 Sterile PBS solution:

[0041] Dissolve 40 g of PBS instant premixed granules in 2 L of deionized water, sterilize at 121°C by autoclaving for 20 min, and store in a refrigerator at 4°C after cooling.

[0042] 2. Design and screening of ASOs targeting ftsZ

[0043] The ftsZ sequences of S. aureus (ATCC29213) and MRSA (USA300 LAC) were searched in GENBANK. After BLAST comparison, the results were as follows: Figure 2 As shown, RNA structure 6.4 was used to predict the secondary structure of ftsZ mRNA, and a series of possible ASOs were designed and synthesized as shown in Table 2. Then, the optimal ASO sequence was obtained based on the bacterial growth curve experiment.

[0044] Table 2

[0045]

[0046] 3. Filter the results.

[0047] ftsZ plays an indispensable role in the division process of bacteria and is an essential protein for bacterial growth. Homology analysis was performed on it in different S. aureus strains by BLAST. Figure 1 As shown in Table 1, it is shown that ftsZ can be used as a suitable target gene for designing ASOs. According to the secondary structure of ftsZ mRNA and the principle of minimum free energy, the seven antisense sequences described in Table 2 were designed. According to the bacterial growth curve experiment, Figure 3 Schematic diagram of the growth curves of different sequences shown, according to which it can be seen that the antisense sequence AS0515 (TTGTCAACGA) shows the best antisense antibacterial activity.

[0048] In one embodiment, the characterization of DP-AD7 is disclosed, as follows:

[0049] 1. Particle size and potential of DP-AD7.

[0050] 1 mL of sample solution was added to the sample dish of the dynamic light scattering (DLS) instrument to detect the particle size and potential of DP-AD7.

[0051] Prepare 1 mL of 20 μM DP-AD7 under the above conditions. Add a drop of DP-AD7 to the copper grid and let it settle for 5 minutes at room temperature. After careful absorption with filter paper, evaporate at room temperature for 3 minutes. The nanoparticle morphology is 10 5 ×, observed under a transmission electron microscope (TEM) at 80 kV.

[0052] 2. Results.

[0053] DLS was used to measure the particle size and potential of nanoparticles. Figure 4 As shown in the figure, the horizontal axis represents the particle size and the vertical axis represents the frequency. The results show that the particle size of DP-AD7 is less than 150nm, the PDI (polydispersity index) is less than 0.4, and the zeta potential is between 14.5mV and 17.3mV. Figure 5 As shown, TEM results further showed that DP-AD7 was approximately spherical with a diameter of less than 100 nm.

[0054] In one embodiment, the delivery efficiency of DP-AD7 to AS0515 in bacteria was tested.

[0055] 1. Flow cytometry was used to determine the delivery efficiency of DP-AD7 in bacteria.

[0056] First, 300 μL of DP-AD7 labeled with FAM AS0515 was prepared. Next, S. aureus (ATCC29213) and MRSA (USA300 LAC) were cultured overnight in TSB and the bacterial concentration was adjusted to 10 using MH medium.6 CFU / mL. Then, 300 μL of different bacterial solutions were added to DP-AD7 to a final concentration of 1 μM, mixed and incubated in a 37°C constant temperature incubator for 1 h. After the incubation, centrifuged at 5000g×5min in a low-temperature centrifuge, carefully discarded the supernatant, washed with 300 μL of sterile PBS, centrifuged again and resuspended with 100 μL of sterile PBS, and the uptake of DP-AD7 nanoparticles by bacteria was detected using the BL 1 channel (green) of the flow analyzer and data analysis was performed using FlowJo.

[0057] 2. Ultra-high resolution microscopic imaging system was used to measure the bacterial uptake by DP-AD7.

[0058] Prepare 300 μL of 0.5 μM DP-AD7 labeled with FAM. Adjust the bacterial concentration of S. aureus (ATCC 29213) and MRSA (USA300 LAC) to 10 μL using MH broth. 8 CFU / mL, take 100μL of bacterial solution and mix with DP-AD7 solution, incubate at 37℃ in the dark for 30min. Centrifuge at 5000g for 5min, wash with 500μL PBS and resuspend twice, resuspend with 100μL PBS. Add 5μL FM4-64 (200μg / mL), mix and stain on ice for 1min. Centrifuge and wash again with the same steps, and finally resuspend with 50μL PBS. Take 10μL of bacterial solution and add it to the center of the slide, evaporate naturally, then add 4μL of anti-fluorescence quencher to seal the slide, cover with a coverslip, and observe the bacterial uptake of DP-AD7 nanoparticles under an ultra-high resolution microscope (Elyra 7).

[0059] 3. Results.

[0060] The results of flow cytometry showed that after incubation with DP-AD7, Figure 6 As shown in the figure, the positive rates of S. aureus (ATCC29213) and MRSA (USA300 LAC) were both greater than 90%, which was basically equivalent to the delivery efficiency (i.e., the fluorescence intensity of the FITC channel) of Lipofectamine2000 (i.e., LF2000 in the figure). To further confirm the delivery ability of DP-AD7, real-time imaging was performed using an Elyra 7 microscope with a 63× / 1.46 oil lens. The acquired images were processed by Zeiss Zen Black software. Figure 7 As shown, after incubation with FAM-labeled DP-AD7 in the dark for 0.5 h, strong green fluorescence was observed in the cytoplasm of both bacteria. Therefore, it can be seen that DP-AD7 can effectively deliver AS0515 to the test strain.

[0061] The present invention also discloses an application of the antisense oligonucleotide sequence in the preparation of a drug for inhibiting Staphylococcus aureus. Specifically, the antisense oligonucleotide sequence is used to inhibit the expression of the ftsZ gene of Staphylococcus aureus.

[0062] In one embodiment, the present invention also conducted an in vitro antibacterial activity experiment of AS0515.

[0063] 1. Preparation of antibiotic solution:

[0064] Oxacillin: Accurately weigh 2.56 mg of oxacillin powder and dilute it to 1024 μg / mL with dd H2O as a mother solution for later use. Then dilute the mother solution to 0.5 μg / mL and store it in a 4°C refrigerator for later use.

[0065] 2. Bacterial growth curve experiment.

[0066] Prepare 300 μL DP-AD and adjust the bacterial concentration of S. aureus (ATCC 29213) and MRSA (USA300 LAC) to 10 in MH broth. 8 CFU / mL, take 30μL of bacterial solution and mix with DP-AD7 and 300μL TSB nutrient broth medium respectively. The normal bacterial growth group was used as the blank control, and oxacillin (0.5μg / mL) was used as the positive control. Take a 100-well culture plate dedicated to the growth curve instrument, take 600μL of bacterial solution and evenly add it to 3 sub-wells, culture at 37℃ for 18-20h, measure the absorbance value at OD600 nm every hour, and obtain the growth curve of different bacteria.

[0067] 3.qRT-PCR experiment.

[0068] According to the ftsZ gene sequence and 16s rRNA of Staphylococcus aureus reported in GENBANK, primers were designed and synthesized by Sangon Biotechnology Co., Ltd., as shown in Table 4.

[0069] Table 4

[0070]

[0071] Bacteria treatment: 30 μL of 10 8 After the two bacterial solutions were incubated with 300 μL DP-AD7 at 37°C for 4 h, they were centrifuged at 12000 g for 5 min, the supernatant was discarded, and the bacterial solution was resuspended in 50 μL dd H2O for later use.

[0072] Trizol method for extracting bacterial RNA: add 10 μL (50 mg / mL) lysozyme to the resuspended bacterial solution, then add 90 μL ddH2O to fully resuspend and mix the bacteria, incubate at 37°C for 30 min, and remix every 5 min. Add 400 μL Trizol, shake vigorously, and then stand at room temperature for 10 min; then add 200 μL chloroform, shake vigorously, emulsify the mixture into a white state, stand at room temperature for 5 min, centrifuge at 12000g for 15 min, take 300 μL of the upper colorless supernatant to another centrifuge tube without RNA / DNA enzyme, then add 400 μL isopropanol, mix, stand at 4°C for 10 min, centrifuge at 12000g for 10 min, and carefully discard the supernatant; then add 1.2 mL of 75% ethanol to resuspend the precipitate, centrifuge at 7500g for 5 min, and discard the supernatant. Open the cap of the centrifuge tube, place it in a clean bench to evaporate, add 30 μL of DEPC water to dissolve the RNA, and store it at -80°C.

[0073] Total RNA quantification: Take 1 μL of RNA solution and place it on the surface of the measuring dish, cover it with the lid, and record the RNA concentration and A260 / A280 ratio.

[0074] RNA reverse transcription: The reverse transcription system was prepared according to the reagent instructions as shown in Table 5 below.

[0075] Table 5

[0076]

[0077] Place 20 μL of the reaction system on a 48-well gradient PCR instrument for reverse transcription reaction under the following reaction conditions: 37°C for 15 min, 85°C for 30 s. Place the resulting cDNA solution on ice for later use, and store the remainder at -80°C.

[0078] RT-PCR: The reaction system was 20 μL (prepared on ice), as shown in Table 6 below.

[0079] Table 6

[0080]

[0081]

[0082] The reaction system was added to a 0.2 ml transparent eight-tube strip and fluorescent quantitative PCR reaction was performed on an RT-PCR instrument. The reaction conditions and time were shown in Table 7.

[0083] Table 7

[0084]

[0085] Melting curve determination conditions were: 95℃ reaction for 15s, 62℃ annealing for 60s, and 95℃ reaction for 15s. Ct values ​​were derived and used as 2 -ΔΔCt Relative quantification was performed by the method.

[0086] 3. Results.

[0087] In order to verify the in vitro antibacterial effect of AS0515, different concentrations of DP-AD7 were prepared to measure its growth inhibition against S. aureus (ATCC29213) and MRSA (USA300 LAC). Figure 8 As shown, the results show that DP-AD7 can effectively inhibit the growth of S. aureus (ATCC29213) in a concentration-dependent manner, where DP-AD7 0.5μM and DP-AD7 1μM respectively represent the inhibition of bacterial growth by DP-AD7 at different concentrations, Mis-DP-AD7 0.5μM represents the inhibition of bacterial growth by the Mismatch (Mis) sequence in Table 2, Oxacillin 0.5μg / ml represents the inhibition of bacterial growth by oxacillin, Free ASOs 1μM represents the inhibition of bacterial growth by only the AS0515 sequence, and Control represents the blank group. Fig. 9 As shown, DP-AD7 has lower activity against MRSA than S. aureus (ATCC29213), but can still completely inhibit bacterial growth when its concentration is 4 μM.

[0088] To further confirm the antibacterial activity of DP-AD7, qRT-PCR was used to detect the inhibition of DP-AD7 on the expression of ftsZ gene of S. aureus (ATCC29213) and MRSA (USA300 LAC). Fig.10 and Fig.11 As shown, the results showed that DP-AD7-encapsulated AS0515 exhibited significant antibacterial activity in both S. aureus (ATCC29213) and MRSA (USA300 LAC), which was basically consistent with the growth curve results. In summary, AS0515 has good in vitro antibacterial activity.

[0089] In summary, the antisense sequence AS0515 (TTGTCAACGA) has the best antibacterial activity, and DP-AD7 is prepared to target ftsZ of Gram-positive bacteria and their resistant bacteria, and obtain good delivery efficiency, antibacterial effect and low cytotoxicity. The present invention interferes with the expression of ftsZ, a gene essential for bacterial survival, to cope with infection by Staphylococcus aureus and its resistant bacteria, and has the advantages of clear target, clear antibacterial activity and low toxicity.

[0090] The present invention also discloses a drug for inhibiting Staphylococcus aureus, which includes the above antisense oligonucleotide sequence. Compared with traditional antibiotic treatment, antisense antibacterial drugs have the advantages of clear targets, low cytotoxicity, and relatively fixed research and development models, making it possible to quickly respond to newly detected drug-resistant strains and effectively avoid the problem of antibiotic resistance.

Claims

1. An antisense oligonucleotide sequence targeting the ftsZ gene of Gram-positive bacteria, characterized in that: The antisense oligonucleotide sequence is: 5'-TTGTCAACGA-3'.

2. Use of the antisense oligonucleotide sequence as claimed in claim 1 in the preparation of drugs for inhibiting Staphylococcus aureus and its drug-resistant bacteria.

3. The use according to claim 2, characterized in that The antisense oligonucleotide sequence of claim 1 is used to inhibit the expression of the ftsZ gene of Staphylococcus aureus and its drug-resistant bacteria.

4. A drug for inhibiting Staphylococcus aureus and its drug-resistant bacteria, characterized in that: Comprising the antisense oligonucleotide sequence of claim 1.

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