Aptamer pila-2 of pseudomonas aeruginosa type iv pilin pila and uses thereof

Abstract

The application belongs to the field of biochemistry and medical microbiological technology, and relates to aptamer PilA-2 of type IV pilus protein PilA of Pseudomonas aeruginosa and use thereof. The application takes the outer segment protein PilA of type IV pilus of Pseudomonas aeruginosa as a target, uses SELEX technology to screen a nucleic acid aptamer specifically combined with the target through 12 rounds, analyzes the base sequence of the aptamer through large-throughput sequencing analysis, obtains the sequence of the nucleic acid aptamer PilA-2 of the type IV pilus protein PilA of Pseudomonas aeruginosa, the nucleic acid sequence of which is PilA-2: 5'-CGTCACACAGCTTACCCCCCGAACATCTCGGTTGC-3'. The base sequence obtained is analyzed through software, the secondary structure of the aptamer is simulated, the affinity size is analyzed, and the influence of the aptamer PilA-2 on the formation of the bacterial membrane of Pseudomonas aeruginosa is explored.

Description

Technical Field

[0001] This invention belongs to the field of medical microbiology and molecular biology technology, and relates to the aptamer PilA-2 of Pseudomonas aeruginosa type IV fimbriae protein PilA and its uses. Background Technology

[0002] Pseudomonas aeruginosa (PA), also known as Pseudomonas aeruginosa, produces pyocyanin and fluorescein, exhibiting a blue-green fluorescence on culture media and forming green pus when infecting wounds. Pseudomonas aeruginosa is an important opportunistic pathogen and one of the most common hospital pathogens, causing a series of serious purulent infections. It exists in the human intestines, skin, and respiratory tract. Infection with Pseudomonas aeruginosa is more likely to occur when a person's immunity is weakened or their defense mechanisms are compromised, or in patients after treatment, procedures, or surgery. It ranks first among non-fermenting bacterial infections. With the widespread use of antibiotics, drug resistance in Pseudomonas aeruginosa is becoming increasingly serious and is showing a year-on-year upward trend, posing significant challenges to clinical treatment and leading to high mortality rates. Currently, in-depth research is being conducted both domestically and internationally on the drug resistance mechanisms of Pseudomonas aeruginosa. Studies have shown that the drug resistance mechanisms of Pseudomonas aeruginosa are exceptionally complex, with the easy formation of biofilms in vivo being one of the main reasons for its resistance. Pseudomonas aeruginosa biofilms are formed when planktonic bacteria adhere to solid surfaces, forming small colonies. The development of tertiary structures and the entire community's coating by bacterial surface polysaccharides further enhance the biofilm's protective function. These biofilms, formed by planktonic bacteria attaching to surfaces and encapsulating bacteria within a polymeric matrix, act as a barrier, allowing them to evade the body's immune response and the killing effects of antibiotics. Furthermore, they undergo significant changes in cell morphology and physiology. Therefore, inhibiting biofilm formation is of great importance.

[0003] *Pseudomonas aeruginosa* possesses type IV pili with polar filaments, which participate in attachment to objects and host tissue surfaces through twitching motion. Twitching motion mediated by pili extension and contraction is essential for *P. aeruginosa* biofilm formation. PilA, a protein in the upper segment of type IV pili, is composed of thousands of subunits that rapidly polymerize and depolymerize via a complex assembly mechanism. It shares a similar lollipop-like topology with small pilins, featuring a highly conserved hydrophobic N-terminal α-helix and a variable C-terminal antiparallel β-sheet. The first 24 residues of the mature pili protein anchor the subunits to the inner membrane until they are polymerized by the assembly mechanism. Playing a crucial role in the twitching motion and adhesion of planktonic bacteria, PilA is thus one of the key proteins in biofilm formation. Studies have shown that the PilA protein in the middle segment of type IV pili plays a key role in bacterial drug resistance. Investigating the form and structure of the PilA protein will contribute to a deeper understanding of the biofilm drug resistance mechanisms of *P. aeruginosa*, providing a basis for bacterial drug resistance mechanisms and interventions. Therefore, altering the structure of type IV fimbriae protein PilA is an effective way to inhibit the formation of biofilms in Pseudomonas aeruginosa.

[0004] Currently, aptamer research is a hot topic. Aptamers are a class of single-stranded nucleic acids (DNA or RNA) capable of binding with high specificity and high affinity to many target molecules (drugs, proteins, inorganic or organic molecules). The systematic evolution of ligands by exponential enrichment (SELEX) system is used to screen for aptamers with high specificity and high affinity. Due to their stable structure and easily modifiable groups, aptamers are frequently used in medical research for targeted therapy and detection. Following the approval of Macugen, an aptamer targeting vascular endothelial growth factor (VEGF), for the treatment of age-related macular degeneration (AMD) in 2004, two other aptamer drugs for the treatment of eye-related diseases—Zimura and Fovista—have entered phase II and phase III clinical trials, respectively (Huang YF, Shangguan DH, Liu HP, Phillips JA, Zhang XL, Chen Y, Tan WH. Molecular Assembly of an Aptamer-Drug Conjugate for Targeted Drug Delivery to Tumor Cells. Chembiochem 2009, 10(5):862-868.). Several other aptamers are currently in different stages of clinical trials, and various medical experiments are underway. However, to date, no aptamer targeting the PilA protein of Pseudomonas aeruginosa type IV has been reported or disclosed. Therefore, this invention intends to consider the application of nucleic acid aptamers to alter the structure of the PilA protein to effectively inhibit the twitching movement and adhesion properties of the pili (the principle is as follows).Figure 1 (As shown). Developing a nucleic acid aptamer targeting type IV fimbriae protein PilA will provide strong support for inhibiting Pseudomonas aeruginosa biofilm formation, intervening in its multidrug resistance, and lay the foundation for targeted therapy and detection of Pseudomonas aeruginosa, which has important clinical value. Summary of the Invention

[0005] The purpose of this invention is to provide the sequence of the nucleic acid aptamer PilA-2, which targets the Pseudomonas aeruginosa IV fimbriae protein PilA.

[0006] A further object of the present invention is to provide the use of the nucleic acid aptamer sequence as a nucleic acid drug that inhibits the formation of Pseudomonas aeruginosa bacterial membranes, and to design targeted treatment regimens for Pseudomonas aeruginosa based on the nucleic acid sequence.

[0007] The method for preparing the PilA-2 sequence of the PilA fimbrial protein PilA aptamer of Pseudomonas aeruginosa IV is as follows:

[0008] The aptamer for Pseudomonas aeruginosa type IV fimbriae protein PilA is a nucleic acid aptamer sequence that targets PilA, a protein in the upper segment of Pseudomonas aeruginosa type IV fimbriae. Its nucleic acid sequence is: PilA-2: 5'-CGTCACACAGCTTACCCCCCGAACATCTCGGTTGC-3'.

[0009] As a preferred application, the PilA-2 aptamer of Pseudomonas aeruginosa IV fimbriae protein PilA is used in the targeted therapy of Pseudomonas aeruginosa infection.

[0010] Preferably, the application of the Pseudomonas aeruginosa IV fimbriae protein PilA aptamer PilA-2 in inhibiting the biofilm of Pseudomonas aeruginosa.

[0011] Preferably, the use of the Pseudomonas aeruginosa IV fimbriae protein PilA aptamer PilA-2 in the preparation of pharmaceuticals or other products.

[0012] Preferably, the drug is a biofilm inhibitor or a suppressor.

[0013] Preferably, the use of the Pseudomonas aeruginosa IV fimbriae protein PilA aptamer PilA-2 in the preparation of probes for detecting Pseudomonas aeruginosa is preferred.

[0014] Preferably, the use of the Pseudomonas aeruginosa IV fimbriae protein PilA aptamer PilA-2 in the preparation of drug targets for detecting Pseudomonas aeruginosa is preferred.

[0015] First, the purified protein PilA was purchased from Shanghai Sangon Biotech. Then, using the SELEX in vitro screening technique for aptamers, the purified PilA protein was collected as a target from a random 78-base ssDNA library (5'-GGGAGCTCAGAATAAACGCTCAA-N). 35 Nucleic acid aptamers specifically binding to the PilA protein were screened from the sample (-TTCGACATGAGGCCCGGATC-3'). The selected sequences were amplified using upstream primer P1 (5'-GGGAGCTCAGAATAAACGCTCAA-3') and phosphate-labeled downstream primer P2 (5'-PO4-GATCCGGGCCTCATGTCGAA-3'). The amplified sequences were then digested into ssDNA for the next round of library selection. After 12 rounds of screening, the samples were sent to Shanghai Sangon Biotech for high-throughput sequencing. The PilA-2 sequence showed a repetition rate of 23.34%, indicating a high frequency of occurrence in the sequencing samples and representing a dominant sequence retained after 12 rounds of screening.

[0016] PilA-2: CGTCACACAGCTTACCCCCCGAACATCTCGGTTGC.

[0017] Then, the aptamer sequence was synthesized, its affinity was analyzed, and the inhibitory effect of different concentrations of aptamer (0, 0.25, 0.5, 1.0, 2.5, 5.0 μM) on bacterial film was investigated.

[0018] In this invention, the aptamer composed of the nucleic acid sequence can specifically bind to the PilA fimbriae protein of Pseudomonas aeruginosa IV, and can be used to inhibit the biofilm of Pseudomonas aeruginosa and for targeted therapy. The aptamer can also serve as a probe or target for detecting the PilA protein.

[0019] Compared with existing related technologies, the present invention has the following advantages:

[0020] 1. This invention obtains the nucleic acid aptamer sequence PilA-2 targeting the Pseudomonas aeruginosa IV fimbriae protein PilA, and its nucleic acid sequences are: 5'-CGTCACACAGCTTACCCCCCGAACATCTCGGTTGC-3'.

[0021] 2. The PilA-2 sequence of the PilA fimbriae IV fimbriae protein aptamer of the present invention plays a significant role in the preparation of drugs or other products.

[0022] 3. The drug containing the PilA-2 sequence of the PilA aptamer of Pseudomonas aeruginosa IV fimbriae provides strong support for biofilm inhibitors and targeted therapy of Pseudomonas aeruginosa, and will have important clinical value. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Schematic diagram illustrating the principle of aptamers inhibiting biofilm formation;

[0025] Figure 2 Aptamer affinity test results;

[0026] Figure 3 Fluorescence inverted microscope observation of aptamers inhibiting bacterial film formation;

[0027] Figure 4 Scanning electron microscopy was used to observe the inhibition of bacterial film formation by aptamers. Detailed Implementation

[0028] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0029] To facilitate a better understanding of the application of this invention, the invention will be further explained below with reference to the accompanying drawings, which show the apparatus of this invention.

[0030] Example 1: Nucleic Acid Aptamer Screening

[0031] The initial screening library was designed as a 78-base random ssDNA library, with primer binding sites of 23 and 20 bases at each end, and a 35-base random sequence in the middle. The library capacity was approximately 435. The library sequence was 5'-GGGAGCTCAGAATAAACGCTCAA-N 35 -TTCGACATGAGGCCCGGATC-3'. PCR primers for the screening process were also designed, including upstream primer P1: 5'-GGGAGCTCAGAATAAACGCTCAA-3' and phosphate-labeled downstream primer P2: 5'-PO4-GATCCGGGCCTCATGTCGAA-3'. These were used for the preparation of ssDNA secondary libraries. All the libraries and primers were synthesized and purified by Shanghai Bioengineering Technology Service Co., Ltd.

[0032] 1. Preparation of various solutions during the screening process

[0033] (1) Protein coating solution: 0.05 mol / L carbonate buffer, Na2CO3 1.59 g, NaHCO3 2.93 g, pH 9.6, stored at 4℃ (store for one month).

[0034] (2) Electrophoresis buffer (5×TBE buffer): Tris 5.4g, boric acid 2.75g, 2ml 0.5M EDTA solution, add double distilled water to dissolve and bring the volume to 100ml, adjust the pH to 8.0 and store at 4℃ for later use.

[0035] (3) 2.5% agarose gel: Add 1.25g agarose powder to 50ml 1×TBE, boil and dissolve in microwave, remove and cool to room temperature at about 45℃, add 2μl ethidium bromide and ethidium bromide, stir evenly and pour into gel, and use after the gel solidifies.

[0036] (4) 1×SELEX binding buffer: 20 mmol / L HEPES (pH 7.35), 120 mmol / L NaCl, 1 mmol / L CaCl2, 5 mmol / L KCl, 1 mmol / L MgCl2, pH 7.35. Weigh 0.4766 g HEPES, 0.7013 g NaCl, 0.0373 g KCl, 0.0147 g CaCl2·2H2O and 0.0203 g MgCl2·6H2O respectively and dissolve them in 80 mL of sterile pure water. Adjust the pH to 7.35, and make up to 100 mL in a volumetric flask. Store at 4 °C.

[0037] (5) PBS buffer (pH 7.4): Weigh 8g NaCl, 0.2g KCl, 0.24g KH2PO4, and 3.63g Na2HPO4˙12H2O, and bring the volume to 1000ml with ultrapure water. Adjust the pH to 7.4. Autoclave in an autoclave for 20min and store at room temperature for later use.

[0038] (6) SELEX washing buffer: binding buffer + 0.05% Tween 20.

[0039] (7) SELEX elution buffer: 20 mmol / L Tris-HCl (pH 8.3), 1 mmol / L DTT, 4 mol / L guanidine isothiocyanate.

[0040] 2. Screening and high-throughput sequencing of nucleic acid aptamers

[0041] (1) Prepare 8 wells of an ELISA plate, add 200 μl of protein coating buffer, then add PilA protein (10 μg for the first round), seal with sealing film, and coat at 4°C overnight or at 37°C for two hours. Prepare another 8 wells of an ELISA plate (reverse screening or control group), add 200 μl of 3% BSA solution, seal with sealing film, and coat at 4°C overnight or at 37°C for two hours.

[0042] (2) Discard the coating solution from the 8 wells containing PilA protein, wash with PBS 2-5 times, pat dry, add 200 μl of 3% BSA solution, seal with sealing film, and incubate in a 37°C water bath for 120 min. (Use BSA to block the sites in the wells where PilA has not bound).

[0043] (3) Dissolve the ssDNA library (2 μg per well for the first round) in 200 μl of SELEX binding buffer, denature at 95°C for 5 min, and then immediately place on ice for 10 min to preserve the library in single-stranded state. (8 wells, i.e., 16 μg, 1600 μl of SELEX binding buffer)

[0044] (4) The 8 wells of the control group (reverse screening) were washed 4 times with PBS. The ssDNA that maintained the single-stranded state was added to the PBS-washed microplate, 200 μL per well, and incubated at 37°C for 30-60 min.

[0045] (5) Wash the 8 wells of the experimental group (immobilized PilA protein) with PBS 2-5 times. Transfer the liquid from the microplate wells after incubation in step (4) to the wells of the experimental group microplate. Seal the wells with sealing film to prevent moisture evaporation. Use 200 μl per well and incubate at 37°C for 30-60 min. Aspirate the liquid from the previous round and store it in an EP tube in case of failure.

[0046] (6) Discard the supernatant after incubation (collect the supernatant in an EP tube to prevent the experiment from failing and you can start incubation again from here). Wash 2-5 times with SELEX washing buffer.

[0047] (7) After patting dry, add 200 μl of SELEX elution buffer and heat at 95°C for 10 min. Transfer the supernatant to a clean EP tube using a pipette. (Add phenol, isoamyl alcohol and chloroform complex to remove protein. Take the supernatant).

[0048] (8) Add 3 times the volume of anhydrous ethanol (pre-cooled) and 1 / 10 volume of sodium acetate (3mol / L NaAC) to the supernatant and place at -80℃ for 40 min (or at -20℃ for more than 30 minutes), then centrifuge at 12000r / min for 10 min.

[0049] (9) Remove the supernatant, add pre-cooled anhydrous ethanol to the precipitate, wash, centrifuge at 12000 r / min for 10 min, remove the supernatant, repeat the above steps once, and leave at room temperature for 10 min after opening the lid.

[0050] (10) After the ethanol has completely evaporated, add 20 μL of TE buffer (sterile) to dissolve the DNA precipitate as the template for the next round of PCR.

[0051] (11) The aptamers obtained in the previous round of screening were used as PCR template DNA, and PCR amplification was performed to obtain an enhanced secondary library. The primers used for PCR were upstream primer P1 and phosphate-labeled downstream primer P2.

[0052] (12) Digest the PCR product with λ enzyme. Calculate the amount of enzyme and double-stranded DNA according to the enzyme's instructions, degrade the phosphate-containing single strand, and obtain an ssDNA secondary library.

[0053] (13) The concentration of the first-round library was determined by measuring the ssDNA using a UV spectrophotometer.

[0054] (14) The libraries selected in the 12th round of screening were sent to Shanghai Sangon Biotech for high-throughput sequencing. High-throughput sequencing yielded 6856 sequences, with each sequence exhibiting repetitions ranging from one to tens of thousands. Due to the multiple rounds of screening, the repetition rate of identical sequences was high. This allowed PCR to significantly amplify the major sequences in the aptamer library, reducing complexity. The PilA-2 sequence had a repetition rate of 23.34%, indicating that the sequence had a high repetition rate in the sequencing samples and was a dominant sequence retained from the 12 rounds of screening. PilA-2: CGTCACACAGCTTACCCCCCGAACATCTCGGTTGC.

[0055] Example 2: Nucleic Acid Aptamer Affinity Analysis

[0056] Aptamer PilA-2 was FAM-labeled, and 0-120 nM concentrations of aptamer were added to 96-well plates coated with PilA protein, and incubated at 37°C for 40 min. Fluorescence values ​​were measured using a fluorescence spectrometer, and affinity analysis was performed using the Kd value calculation formula.

[0057] The affinity test results of the aptamers are as follows: Figure 2 As shown, the fluorescence value is directly proportional to the concentration of the aptamer within a certain range, indicating that the aptamer has a high affinity for the PilA protein. Affinity performance analysis using the formula yields the Kx of the aptamer for the PilA protein. d Value, K of aptamer PilA-2 d The value was 32.26±2.72nM, reaching the nM level, indicating a relatively ideal affinity.

[0058] Example 3: Nucleic acid aptamers inhibit Pseudomonas aeruginosa biofilm formation

[0059] 1. Observe the bacterial film using a fluorescence inverted microscope.

[0060] like Figure 3 As shown, the intervention of aptamer PilA-2 on Pseudomonas aeruginosa bacterial membrane was evaluated visually using a fluorescence inverted microscope.

[0061] (1) Add a clean glass cover glass with a diameter of 20 mm to a 12-well cell culture plate. Take 10⁸ CFU / mL of Pseudomonas aeruginosa and inoculate it at 1% in 1 mL of sterile LB medium. Mix well.

[0062] (2) Add aptamers of different concentration gradients into cell culture well plates and mix (with the final concentrations of the aptamer components in the structure being 0, 0.25, 0.5, 1.0, 2.5, and 5.0 μM, respectively), and incubate at 37°C and 100 r / min on a shaker for 24 h, 48 h, and 72 h, respectively.

[0063] (3) Discard the liquid in the well plate, treat with 1 mL of fixative for 10 min, and under light-shielding conditions, stain the bacterial film with SYTO9 green fluorescent nucleic acid staining agent. Observe the growth of the target bacteria film at different time periods under a fluorescent inverted microscope.

[0064] 2. Scanning electron microscopy observation of aptamers inhibiting biofilm formation.

[0065] like Figure 4 As shown, the intervention of aptamer PilA-2 on Pseudomonas aeruginosa bacterial membrane was visually evaluated using scanning electron microscopy.

[0066] (1) Add a clean glass cover glass with a diameter of 20 mm to a 12-well cell culture plate. Take 10⁸ CFU / mL of Pseudomonas aeruginosa and inoculate it at 1% in 2 mL of sterile LB medium. Mix well.

[0067] (2) Add aptamers of different concentration gradients into cell culture well plates and mix (with the final concentrations of the aptamer components in the structure being 0, 0.25, 0.5, 1.0, 2.5, and 5.0 μM, respectively), and incubate at 37°C and 100 r / min on a shaker for 24 h, 48 h, and 72 h, respectively.

[0068] (3) After the culture is completed, remove the coverslip and rinse it slowly with sterile water 3 times. Let it air dry naturally. Spray the sample with gold and observe the growth of the target bacteria film at different time periods under a scanning electron microscope.

[0069] The present application has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present application. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and implementation methods of the present application without departing from the spirit and scope of the present application, and all such modifications and improvements fall within the scope of the present application. The scope of protection of the present application is determined by the appended claims.

Claims

1. PilA-2, an aptamer for Pseudomonas aeruginosa type IV fimbriae protein PilA, characterized in that: Its nucleic acid sequence is: PilA-2: 5'-CGTCACACAGCTTACCCCCCGAACATCTCGGTTGC-3'.

2. The aptamer PilA-2 for Pseudomonas aeruginosa type IV fimbriae protein PilA as described in claim 1, characterized in that: Application of aptamers in the preparation of drugs for inhibiting Pseudomonas aeruginosa biofilm formation.

3. The aptamer PilA-2 for Pseudomonas aeruginosa type IV fimbriae protein PilA as described in claim 1, characterized in that: The use of aptamers in the preparation of probes for detecting Pseudomonas aeruginosa.

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