Nucleic acid aptamer specifically binding to ompk36 protein and use thereof

Abstract

This invention discloses nucleic acid aptamers that specifically bind to the OmpK36 protein and their applications. Through analysis and screening, aptamers APT-1 and APT-2 that specifically bind to the OmpK36 protein were obtained. Nucleic acid aptamer APT-1 can specifically bind to outer membrane polypeptides, while nucleic acid aptamer APT-2 can specifically bind to polypeptides specific to Klebsiella pneumoniae. Both can bind to the OmpK36 protein to form a complex structure, which can be used for the specific detection of Klebsiella pneumoniae, providing convenience for clinical detection, diagnosis and treatment, and exclusion detection of specific pathogens in SPF animals.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid aptamer technology, specifically to nucleic acid aptamers that specifically bind to the OmpK36 protein and their applications. Background Technology

[0002] Klebsiella pneumoniae, a Gram-negative opportunistic pathogen, is commonly found in the intestinal and respiratory tracts, but can cause multi-system infections such as pneumonia and sepsis under immunodeficiency or environmental stress. Accurate and rapid detection of Klebsiella pneumoniae is of great significance for clinical diagnosis, treatment, and prevention.

[0003] Taking the detection of Klebsiella pneumoniae infection in SPF-grade animals as an example, the detection of Klebsiella pneumoniae infection in SPF-grade animals (specific pathogen-free animals) is a crucial step in ensuring the health of laboratory animals and the reliability of research data. Because SPF animals must be strictly excluded from specific pathogens, their infection monitoring requires highly sensitive and specific methods, mainly including the following three types of techniques:

[0004] 1. Bacterial Culture and Biochemical Identification: Traditional detection methods involve collecting animal throat swabs, feces, or tissue samples and culturing them on blood agar plates or MacConkey broth. Klebsiella pneumoniae forms mucoid colonies on blood agar plates and can be preliminarily identified by biochemical characteristics such as a negative oxidase test and a positive urease test. For example, the national standard GB / T 14926.13-2001 clearly specifies the entire process from sample processing to strain confirmation, including key steps such as the triple sugar iron test and the lysine decarboxylase test. This method is time-consuming, typically requiring 48-72 hours.

[0005] 2. Molecular Biological Detection: To improve detection efficiency, multiplex PCR technology is widely used. For example, specific primers for Klebsiella pneumoniae are designed, and the detection time is only 4-6 hours. Furthermore, real-time quantitative PCR (qPCR) can quantitatively assess bacterial load using Ct values, making it suitable for large-scale population monitoring. Studies have shown that molecular methods have a 30% higher detection rate for asymptomatic carriers than culture methods, especially in the routine monitoring of SPF animal barrier facilities. This method relies on high-quality pre-treated DNA samples.

[0006] 3. Serological and pathological testing: For suspected cases, specific antibodies in animal serum can be detected by enzyme-linked immunosorbent assay (ELISA). This method is limited by the time window for antibody production (usually 7-10 days after infection). Pathological examination focuses on tissue damage characteristics, such as neutrophil infiltration, hemorrhagic necrosis, and microabscess formation in lung tissue sections. The observation of short rod-shaped Gram-negative bacteria in conjunction with Gram staining can aid in diagnosis.

[0007] Aptamers are a class of single-stranded DNA or RNA molecules obtained through in vitro screening techniques. They can bind to target molecules (such as proteins, small molecules, and cells) with high affinity and high specificity through specific three-dimensional structures. Their name originates from the Latin word "aptus" (fitting) and the Greek word "meros" (part), meaning "fitting unit." Aptamers were discovered in 1990, independently developed by Tuerk and Gold's team and Ellington's team. The core technology is SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This technology, through multiple rounds of screening and amplification, enriches nucleic acid molecules that specifically bind to targets from random sequence libraries, laying the foundation for aptamer research. The core advantages of aptamers lie in their small molecular weight (typically 8-15 kDa), high stability (especially DNA aptamers, which are resistant to high temperatures and enzymatic digestion), non-immunogenicity, and ease of chemical modification. Compared to traditional antibodies, aptamers can be synthesized in vitro, avoiding animal dependence, offering better batch-to-batch consistency, and targeting non-immunogenic molecules (such as toxins or small molecules) that are difficult for traditional antibodies to recognize. Furthermore, aptamers can be modularly designed to integrate fluorescent, electrochemical groups, or nanomaterials, expanding their applications in detection and treatment.

[0008] If a nucleic acid aptamer with specific affinity for Klebsiella pneumoniae is identified, it will be of great significance for clinical diagnosis and treatment as well as for the exclusion and detection of specific pathogens in SPF animals. Summary of the Invention

[0009] This invention uses structural analysis of the OmpK36 protein of Klebsiella pneumoniae to screen out two polypeptides exposed on the outer membrane. For each of these two polypeptides, two aptamers with specific affinity are screened using SELEX technology, providing a better selection for the detection of Klebsiella pneumoniae.

[0010] In view of this, the solution of the present invention is as follows:

[0011] The first aspect of the present invention is to provide a nucleic acid aptamer that specifically binds to the OmpK36 protein, which is nucleic acid aptamer APT-1 or APT-2, and the corresponding nucleotide sequences are shown in SEQ ID NO.1 and 2.

[0012] Furthermore, it also includes truncating, extending, mutating, tandemly binding, or circularizing the nucleotide sequences of nucleic acid aptamers APT-1 or APT-2 while still containing key nucleic acid motifs and retaining the nucleotide sequences capable of binding to the OmpK36 protein.

[0013] Furthermore, the nucleic acid aptamer APT-1 is used to specifically bind the polypeptide DIFDGI, and the nucleic acid aptamer APT-2 is used to specifically bind the polypeptide DNSFTRSAGIS.

[0014] A second aspect of the present invention is to provide a kit for detecting Klebsiella pneumoniae, comprising nucleic acid aptamers APT-1 and / or APT-2 that specifically bind to the OmpK36 protein, wherein the nucleotide sequences of said nucleic acid aptamers APT-1 and APT-2 correspond to those shown in SEQ ID NO.1 and 2, respectively.

[0015] Furthermore, the nucleotide sequence of the nucleic acid aptamer contains markers or chemically modified bases.

[0016] Furthermore, the markers include, but are not limited to, any one or more of isotope markers, fluorescent markers, biotin markers, enzyme markers, and chemiluminescent markers; the chemical modifications include, but are not limited to, any one or more of methylation, amination, thiolation, phosphorylation, thiolation, carboxylation, and isotopeation.

[0017] Preferably, the kit further includes cDNA designed to correspond to the nucleic acid aptamer, one end of which is labeled with a fluorescent group, and the cDNA is complementary to the end away from the labeled fluorescent group. One end of the cDNA is also labeled with a quenching group. During detection, when the nucleic acid aptamer binds to Klebsiella pneumoniae, the cDNA is released, and the fluorescent dye moves away from the quenching group. The supernatant is collected by centrifugation, and the fluorescence intensity is measured. An increase in fluorescence intensity indicates detection.

[0018] It is understandable that the above tests can be qualitative or quantitative.

[0019] A third aspect of the present invention is to provide the use of the nucleic acid aptamer described in the first aspect in the preparation of a Klebsiella pneumoniae detection product.

[0020] Furthermore, the products include reagent kits, biosensors, or detection chips.

[0021] Furthermore, the product is used for SPF-grade animal Klebsiella pneumoniae detection.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The nucleic acid aptamers APT-1 or APT-2 provided by this invention can specifically bind to the OmpK36 protein. Specifically, nucleic acid aptamer APT-1 can specifically bind to the outer membrane polypeptide, and nucleic acid aptamer APT-2 can specifically bind to the polypeptide unique to Klebsiella pneumoniae. Both can bind to the OmpK36 protein to form a complex structure, which can be used for the specific detection of Klebsiella pneumoniae, providing convenience for clinical detection and treatment as well as the exclusion detection of specific pathogens in SPF animals. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the OmpK36 protein structure described in this invention.

[0025] Figure 2 This is a secondary structure diagram of the aptamer APT-1 predicted and analyzed in Embodiment 1 of the present invention.

[0026] Figure 3 This is a schematic diagram showing the predicted structure of the complex binding between aptamer APT-1 and OmpK36 protein in Example 1 of the present invention.

[0027] Figure 4 This is a secondary structure diagram of the aptamer APT-2 predicted and analyzed in Embodiment 1 of the present invention.

[0028] Figure 5 This is a schematic diagram of the predicted structure of the aptamer APT-2 and OmpK36 protein binding complex in Example 1 of the present invention.

[0029] Figure 6 This is the fluorescence intensity curve of the binding between aptamer APT-1 and DIFDGI peptide in Example 2 of the present invention.

[0030] Figure 7 This is the fluorescence intensity curve of the binding between the aptamer APT-2 and the DIFDGI peptide in Example 2 of the present invention.

[0031] Figure 8 This is a comparison of the binding fluorescence intensity of aptamer APT-1 in different bacterial species in Example 3 of the present invention.

[0032] Figure 9 This is a comparison of the binding fluorescence intensity of aptamer APT-2 in different bacterial species in Example 3 of the present invention. Detailed Implementation

[0033] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. 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.

[0034] OmpK36 of Klebsiella pneumoniae is an outer membrane porin that spans the entire outer membrane lipid bilayer.

[0035] OmpK36 belongs to the β-barrel porin family. Its structural features include: transmembrane β-strands: typically composed of 16-18 antiparallel β-sheets forming a barrel-like structure; loops: connecting the β-sheets, partially exposed to the outer membrane (environment) and partially extending into the periplasmic space (inner side). As a β-barrel porin, its polypeptide chains form β-sheets, creating a hydrophilic channel that allows passive diffusion of small molecules (such as nutrients and antibiotics). The N-terminus and C-terminus are located on the inner side of the outer membrane (facing the periplasmic space), while the loops are exposed on the outer side of the outer membrane (facing the environment).

[0036] OmpK36's pore size and charge properties control molecular permeability, influencing bacterial sensitivity to antibiotics. As a key component of the outer membrane, it, along with LPS, restricts the entry of hydrophobic molecules, forming a resistance barrier for Gram-negative bacteria. The amino acid sequence of OmpK36 is as follows:

[0037] MKVKVLSLLVPALLVAGAANAAEIYNKDGNKLDLYGKIDGLHYFSDDKDVDGDQTYMRLGVKGETQINDQLTGYGQWEYNVQANNTESSSDQAWTRLAFAGLKFGDAGSFDYGRNYGVVYDVTSWTDVLPEFGGDTYGSDNFLQSRANGVATYRNSDFFGLVDGLNFALQYQGKNGSVSGE GATNNGRGALKQNGDGFGTSVTYDIFDGISAGFAYANSKRTDDQNQLLLGEGDHAETYTGGLKYDANNIYLATQYTQTYNATRAGSLGFANKAQNFEVAAQYQFDFGLRPSVAYLQSKGKDLNGYGDQDILKYVDVGATYYFNKNMSTYVDYKINLLDDNSFTRSAGISTDDVVALGLVYQF

[0038] Protein structure (1OSM) as follows Figure 1 As shown.

[0039] Example 1: Screening of aptamers

[0040] By analyzing the X-ray crystallography (1OSM) structure of OmpK36, two polypeptide sequences, DIFDGI (P1) and DNSFTRSAGIS (P2), were located on the outer side. The structural formulas of polypeptides P1 and P2 are shown below.

[0041]

[0042]

[0043] 1. Aptamer screening for peptide P1

[0044] For peptide P1, its amino acid sequence is DIFDGI, where D is aspartic acid (negatively charged), I is isoleucine (hydrophobic), and F is phenylalanine (aromatic and hydrophobic). The following D and G are again aspartic and glycine, respectively, with G being more flexible. The last I is isoleucine. By analyzing the physicochemical properties of the target peptide—charge, hydrophobicity, and aromaticity—candidate aptamer sequences are designed, considering complementary charge interactions, hydrophobic interactions, and structural matching. The goal is to ensure that the aptamer can form stable secondary structures, such as hairpins or G tetramers, to promote binding.

[0045] Taking all the above factors into consideration, the aptamer was selected based on capture-SELEX. The following aptamers showed significant affinity for APT-1:

[0046] GGGTTATGGGTGGATAGGGTTTTCGCTATTTCGCAGTAGCGTTTTATCGGTTGGGTTAATCCGAT (SEQID NO.1)

[0047] The secondary structure of aptamer APT-1 was predicted and analyzed using Mfold software, such as... Figure 2 As shown.

[0048] The structure of the aptamer APT-1 binding complex to OmpK36 protein, as predicted by the analysis, is as follows: Figure 3 As shown, the prediction analysis shows that the nucleotides at positions 27-29 of the aptamer form intermolecular bonds with the amino acids at positions 184-189 of the OmpK36 protein.

[0049] 2. Screening of nucleic acid aptamers for peptide P2

[0050] DNSFTRSAGIS is an alpha helix in which D (aspartic acid) is negatively charged, N (asparagine) is polar and may form hydrogen bonds; S (serine) is polar and the hydroxyl group may participate in hydrogen bonding; F (phenylalanine) is hydrophobic and contains an aromatic ring; T (threonine) is polar and contains a hydroxyl group; R (arginine) is positively charged; S (serine) is the same as above; A (alanine) is hydrophobic; G (glycine) is flexible; I (isoleucine) is hydrophobic; S (serine) is the same as above.

[0051] Regarding charge, the presence of D and R implies that the polypeptide may carry regions of both positive and negative charges. Hydrophobic residues F, A, and I may form a hydrophobic core, while glycine (G) provides structural flexibility. Due to the α-helix structure, the periodicity of the helix may allow positively charged R residues to be located on one side of the helix, forming a charge cluster, while hydrophobic residues are located on the other side.

[0052] Taking the above factors into consideration, the selection of aptamers was conducted. Based on the capture-SELEX screening method, the following aptamers with significant affinity for APT-2 were selected:

[0053] GGGCTAGGGCTAGGGCTAGGGTTTTCGCGTTTTTCGCGTTTTATCGATTTATCGAT (SEQ ID NO.2)

[0054] The secondary structure of aptamer APT-1 was predicted and analyzed using Mfold software, such as... Figure 4 As shown.

[0055] The structure of the aptamer APT-2 and OmpK36 protein binding complex, as predicted by HDock, is shown below. Figure 5 As shown, the prediction analysis shows that aptamers 2-4, 14, and 30-32 form intermolecular interaction bonds with amino acids at positions 319-329 of the OmpK36 protein.

[0056] Example 2: Experiment on the binding of aptamers to peptides

[0057] 1. Binding experiment of APT-1 to DIFDGI peptide

[0058] The selected dye ThT emits strong fluorescence with nucleic acid aptamers and competes with peptides for binding sites on the aptamers. The peptides can bind to the nucleic acid aptamers to form stable complexes, altering the aptamer structure and weakening dye binding, thus decreasing fluorescence intensity. Peptide concentration gradients of 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM were set up, and the peptides bound to the Apt-1 aptamer. Fluorescence intensity was observed using a microplate reader, and a blank control was included. Figure 6The results showed that the fluorescence of ThT binding to the nucleic acid aptamer decreased as the concentration of the peptide increased, indicating that the aptamer and peptide have a stoichiometric effect, and the Kd value of the peptide aptamer was 20.61 μM.

[0059] 2. Binding experiment of APT-2 with DNSFTRSAGIS peptide

[0060] The selected dye ThT emits strong fluorescence with nucleic acid aptamers and competes with peptides for binding sites on the aptamers. The peptides can bind to the nucleic acid aptamers to form stable complexes, altering the aptamer structure and weakening dye binding, thus decreasing fluorescence intensity. Peptide concentration gradients of 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM were set up, and the peptides bound to the Apt-2 aptamers. Fluorescence intensity was observed using a microplate reader, and a blank control was included. Figure 7 The results showed that the fluorescence of ThT binding to the nucleic acid aptamer decreased as the concentration of the peptide increased, indicating that the aptamer and peptide have a stoichiometric effect, and the Kd value of the peptide aptamer was 25.31 μM.

[0061] Example 3 Aptamer-bacterial binding assay

[0062] 1. Aptamer APT-1 binds to bacteria

[0063] The P1 polypeptide sequence is relatively short and, although not specific to the OmpK36 protein, only OmpK36 is an outer membrane protein, exhibiting specificity for binding and enrichment of Klebsiella pneumoniae. The aptamer APT-1 sequence was labeled as: GGGTTATGGGTGGATAGGGTTTTCGCTATTTCGCAGTAGCGTTTTATCGGTTGGGTTAATCCGAT-BHQ1, and the corresponding cDNA was labeled as FAM-TGCGAAATAGCG, constructing an aptamer biosensor. When the aptamer binds to Klebsiella pneumoniae, cDNA is released, and the FAM dye moves away from the quenching group BHQ1. After centrifugation, the supernatant was collected, and the fluorescence intensity was measured; the fluorescence intensity increased. Following the above method, the binding ability of the aptamer was compared by setting the same concentration of CFU / ml for Klebsiella pneumoniae, Bacillus subtilis, Escherichia coli, and Bifidobacterium lactis. Using the above method, it was found that the fluorescence intensity of the aptamer APT-1 binding to Klebsiella pneumoniae was significantly higher than that of other types. Figure 8 Furthermore, when the aptamer APT-1 was unavailable, the fluorescence intensity differences among the groups were minimal. This indicates that APT-1 can be used for the specific detection of Klebsiella pneumoniae.

[0064] 2. The binding of aptamer APT-2 to bacteria

[0065] The P2 peptide is specific to Klebsiella pneumoniae. The aptamer APT-2 sequence was labeled as: GGGCTAGGGCTAGGGCTAGGGTTTTCGCGTTTTTCGCGTTTTATCGATTTATCGAT-BHQ1, and the corresponding cDNA was labeled as FAM-ACCCTAGCCCTA. An aptamer biosensor was constructed. When the aptamer binds to Klebsiella pneumoniae, the cDNA is released, and the FAM dye moves away from the quenching group BHQ1. After centrifugation, the supernatant was collected and the fluorescence intensity was measured. The fluorescence intensity increased.

[0066] Following the above method, by setting the same concentration of CFU / ml for Klebsiella pneumoniae, Bacillus subtilis, Escherichia coli, and Bifidobacterium lactis, the binding ability of the aptamers was compared. Using the above method, it was found that the fluorescence intensity of aptamer APT-2 binding to Klebsiella pneumoniae was significantly higher than that of other types. Figure 9 Furthermore, in the absence of the aptamer APT-2, the fluorescence intensity differences among the groups were minimal. This indicates that APT-2 can be used for the specific detection of Klebsiella pneumoniae.

[0067] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A nucleic acid aptamer that specifically binds to the OmpK36 protein, characterized in that, The aptamers are APT-1 or APT-2, and the corresponding nucleotide sequences are shown in SEQ ID NO.1 and 2.

2. The nucleic acid aptamer according to claim 1, characterized in that, It also includes nucleotide sequences that retain key nucleic acid motifs and the ability to bind to OmpK36 protein after being truncated, extended, mutated, tandemly linked, or circularized by the nucleotide sequences of nucleic acid aptamers APT-1 or APT-2.

3. The nucleic acid aptamer according to claim 1 or 2, characterized in that, The nucleic acid aptamer APT-1 is used to specifically bind the polypeptide DIFDGI, and the nucleic acid aptamer APT-2 is used to specifically bind the polypeptide DNSFTRSAGIS.

4. A kit for detecting Klebsiella pneumoniae, characterized in that, The aptamers include APT-1 and / or APT-2, which specifically bind to the OmpK36 protein, and the nucleotide sequences of APT-1 and APT-2 correspond to those shown in SEQ ID NO. 1 and 2, respectively.

5. The reagent kit according to claim 4, characterized in that, The nucleotide sequence of the nucleic acid aptamer contains a marker or the bases are chemically modified.

6. The reagent kit according to claim 5, characterized in that, The marker is selected from at least one of isotope markers, fluorescent markers, biotin markers, enzyme markers, and chemiluminescent markers.

7. The reagent kit according to claim 5, characterized in that, The chemical modification is selected from at least one of methylation, amination, thiolation, phosphorylation, thiolation, carboxylation, and isotopication.

8. The use of the nucleic acid aptamer according to claim 1 in the preparation of Klebsiella pneumoniae detection products.

9. The application according to claim 8, characterized in that, The products include reagent kits, biosensors, or detection chips.

10. The application according to claim 8, characterized in that, The product is used for SPF-grade detection of Klebsiella pneumoniae in animals.

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