Nucleic acid aptamer combined with HRP2 protein and application of nucleic acid aptamer

Abstract

The invention belongs to the technical field of biology, and discloses a nucleic acid aptamer capable of being specifically combined with HRP2 protein and application of the nucleic acid aptamer. Through a systematic chemical modification and screening technology, the nucleic acid aptamer with remarkable advantages is successfully obtained. The nucleic acid aptamer not only has small molecular weight, but also has highly stable chemical properties, and shows good adaptability in the aspects of preservation and marking. From the aspect of structural characteristics, the nucleic acid aptamer has the characteristics of stable structure and simple configuration, so that the nucleic acid aptamer has remarkable advantages in the aspect of chemical modification. Meanwhile, the nucleic acid aptamer can be obtained through artificial synthesis in a short time, the excellent chemical stability of the nucleic acid aptamer provides guarantee for long-term storage, and diversified labeling operation is easy to carry out. Based on the characteristics, the nucleic acid aptamer has wide application potential in the field of biomedicine, can be applied to multiple directions of biomolecular detection, disease diagnosis, living imaging, targeted therapy and the like, and shows wide application prospect and important scientific research value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and specifically to a nucleic acid aptamer that binds to the HRP2 protein and its applications. Background Technology

[0002] Hepatocellular carcinoma-derived growth factor-associated protein 2 (HRP2) is a nuclear protein that finely regulates the expression of downstream target genes. HRP2 belongs to the HDGF protein family and possesses a highly conserved N-terminal PWWP domain, which specifically recognizes and binds to specific modification markers on histones, particularly the trimethylation of histone H3 at position 36 (lysine). HRP2 functions as a transcriptional co-regulator; it does not directly bind to DNA but can activate or inhibit the expression of specific genes through interactions with other transcription factors, RNA polymerase II, or chromatin remodeling complexes.

[0003] As a member of the growth factor family, HRP2 participates in regulating the cell cycle. It promotes cell proliferation and differentiation by regulating a series of genes related to the cell cycle (such as Cyclins, CDKs) and apoptosis (such as the Bcl-2 family). Multiple studies, including "HDGF-related protein-2 (HRP-2) acts as an oncogene to promote cell growth in hepatocellular carcinoma," have confirmed that HRP2 plays a role in cancer development and progression, acting as an oncogene by driving proliferation, inhibiting apoptosis, and promoting metastasis, and is closely related to poor patient prognosis. Therefore, based on its role in tumor development and its multi-mechanism carcinogenic characteristics, further research and exploration of precision treatment strategies targeting HRP2 are warranted.

[0004] Nucleic acid aptamers are single-stranded oligonucleotides, typically 15-60 base pairs long, obtained through screening in vitro (SELEX) technology, consisting of DNA or RNA. They fold into specific three-dimensional structures, binding to target molecules (such as proteins, small molecules, and metal ions) with high specificity and affinity. Compared to small molecule drugs and antibodies, nucleic acid aptamers not only possess comparable or even superior specificity and affinity, but also offer advantages such as shorter in vitro screening cycles, a broader target range, lower production costs, easier large-scale preparation, easier chemical synthesis and modification, and lower immunogenicity. Currently, nucleic acid aptamer production processes are centered around SELEX technology, combined with chemical synthesis, modification, and quality control, achieving a highly efficient and precise production process. Due to their high specificity, strong affinity, low immunogenicity, and flexible modification capabilities, nucleic acid aptamers have demonstrated significant advantages in areas such as targeted cancer therapy, molecular diagnostics, and drug delivery. Due to the numerous significant advantages of nucleic acid aptamers, they have shown broad application prospects in many fields such as biosensor technology, molecular imaging, targeted drug delivery, and tumor treatment. Summary of the Invention

[0005] To overcome the current challenges in HRP2-driven targeted cancer therapy and further enhance the ability to target HRP2, this invention provides a novel nucleic acid aptamer that binds to the HRP2 protein. This aptamer possesses multiple advantages: its small molecular weight facilitates penetration of biological membrane structures; its highly stable chemical properties allow it to maintain activity under various environmental conditions; it is easy to store, requiring no complex low-temperature or special environmental preservation conditions, and its labeling operation is simple. Furthermore, this nucleic acid aptamer exhibits high affinity for the HRP2 protein. Based on these characteristics, this nucleic acid aptamer demonstrates significant application potential in multiple dimensions, including disease detection, clinical diagnosis, molecular imaging, and targeted therapy, and is expected to bring new breakthroughs to the diagnosis and treatment of HRP2-driven cancers and related diseases.

[0006] The technical solution of the present invention is as follows: On one hand, the present invention provides a nucleic acid aptamer for binding HRP2 protein, characterized in that the nucleic acid aptamer comprises a nucleotide sequence having at least 70% homology with the nucleotide sequence shown in SEQ ID NO.1 and binding HRP2 protein; The nucleotide sequence shown in SEQ ID NO.1 is specifically TTCAGCACTCCACGCATAGCTG TACCCGGCTGTACTGTCCGGGATGGTGTCTGAACCCTATGCGTGCTACCGTGAA.

[0007] As a specific embodiment of the present invention, the nucleic acid aptamer includes the nucleotide sequence shown in SEQ ID NO.1.

[0008] As a specific embodiment of the present invention, the nucleic acid aptamer comprises an RNA sequence transcribed from the nucleotide sequence shown in SEQ ID NO.1.

[0009] It is understood that any nucleotide sequence that has at least 70% homology with the nucleic acid aptamer provided by the present invention and binds to the HRP2 protein, for example, by deleting or adding a portion of the nucleotide sequence shown in any of the above-mentioned nucleic acid aptamers, still has a high affinity for the HRP2 protein and is still within the protection scope of the present invention.

[0010] The present invention also provides a conjugate of a nucleic acid aptamer, wherein the conjugate of the nucleic acid aptamer is formed by attaching any substance for labeling, detection, diagnosis or treatment to the nucleotide sequence of the nucleic acid aptamer, and the conjugate of the nucleic acid aptamer after attaching the substance specifically binds to the HRP2 protein.

[0011] As a specific embodiment of the present invention, the substance includes at least one of fluorescent markers, quenching groups, azide groups, alkynyl groups, radioactive substances, therapeutic substances, biotin, digoxin, maleimide, nanomaterials, polyethylene glycol, small peptides, siRNA, and enzymes.

[0012] The fluorescent marker used in this embodiment of the invention is specifically FAM as an example. The modified nucleic acid aptamer sequence should meet specific functional requirements. Specifically, firstly, the binding affinity to the HRP2 protein should reach or exceed the level of the original parent sequence; secondly, if the affinity enhancement is limited, its structural stability should be significantly enhanced to ensure effectiveness and reliability in practical applications.

[0013] In other words, the modified nucleic acid aptamers described above have molecular structures, physicochemical properties, and functions that are basically the same as or similar to the original nucleic acid sequences, and can be used to bind to the HRP2 protein.

[0014] In another aspect, the present invention provides applications of the nucleic acid aptamer, conjugates of the nucleic acid aptamer, or derivatives of the nucleic acid aptamer, selected from any of the following: 1) Purification or detection of HRP2 protein for non-diagnostic purposes; 2) Localization imaging of cells, tissues, or in vivo expressing HRP2 protein for non-diagnostic purposes; 3) As an inhibitor of HRP2 protein; 4) Preparation of drugs targeting the HRP2 protein; The amino acid sequence of the HRP2 protein is shown in SEQ ID NO.2; specifically: GSVEEKLQKLHS EIKFALKVGSPDVKRCLNALEELGTLQVTSQILQKNTDVVATLKKIRRYKANKDVMEKAAEVYTRLKSR.

[0015] In another aspect, the present invention provides the use of the nucleic acid aptamer, the conjugate of the nucleic acid aptamer, or the derivative of the nucleic acid aptamer in the preparation of a drug for targeting tumors.

[0016] As a specific embodiment of the present invention, the tumor includes liver cancer and / or colorectal cancer.

[0017] In another aspect, the present invention provides a drug targeting the HRP2 protein, comprising the nucleic acid aptamer, a conjugate of the nucleic acid aptamer, or a derivative of the nucleic acid aptamer.

[0018] Beneficial effects of the present invention 1. Screening to obtain a nucleic acid aptamer with small molecular weight, stable chemical properties, easy storage and labeling, which can bind to HRP2 protein with high affinity and high specificity; 3. The structure is relatively stable, simple, easy to modify, and can be artificially synthesized in a short period of time; it is chemically stable, easy to store, and easy to label. 4. It can be used in detection, diagnosis, imaging and treatment, such as for the purification or high-sensitivity detection of HRP2 protein; it can be used to prepare drugs targeting HRP2 protein, etc., with broad application prospects. Attached Figure Description

[0019] Figure 1 For the prediction of the secondary structure of the nucleic acid aptamer HiApt; Figure 2 A schematic diagram illustrating the affinity of the nucleic acid aptamer HiApt to the HRP2 protein for FP validation. Figure 3 A schematic diagram showing the results of flow cytometry analysis of the affinity between the nucleic acid aptamer HiApt and the HRP2 protein; Detailed Implementation

[0020] The present invention will be further described in detail below with reference to embodiments. It should be noted that the embodiments described below are intended to facilitate understanding of the present invention and are not intended to limit it in any way. Reagents not specifically mentioned in this embodiment are all known products obtained by purchasing commercially available products.

[0021] Example 1: Screening of HRP2-binding ssDNA aptamers The method for screening ssDNA aptamers that bind to the HRP2 protein includes the following steps: 1. Expression and purification of HRP2 protein The constructed HRP2 plasmid was transformed into BL21(DE3) competent cells. Single clones were picked from kanamycin-resistant plates and cultured in LB medium at 37°C with a final concentration of 0.05 mg / mL kanamycin. When the OD value reached 0.8–1, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and the cells were induced overnight at 16°C. After centrifugation to collect the cell pellet, the cells were resuspended in DPBS buffer (2.5 mM potassium chloride, 1.5 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 8 mM disodium hydrogen phosphate dodecahydrate; pH 7.5). Under low temperature conditions, imidazole was added to the resuspending solution, followed by sonication, high-speed centrifugation to remove cell debris, and collection of the supernatant. The supernatant was slowly added to a Ni-NTA Beads gravity column. The Ni column was washed with 15 to 20 column volumes of Washing Buffer (DPBS buffer containing 10 mM, 40 mM imidazole, and 2 M NaCl). Finally, the column was eluted with 5 to 10 column volumes of Elution Buffer (DPBS buffer containing 300 mM imidazole and 10% glycerol), and the eluent was collected. The eluent and TEV (for removing the His-Tag) were placed in a dialysis bag and dialyzed overnight at 4°C in dialysis buffer (DPBS buffer containing 10% glycerol). The dialyzed protein sample was added back to the Ni-NTA Beads gravity column and flow-through was repeated 3-4 times. The flow-through was collected, and the protein was concentrated to a concentration of 20 mg / mL using a 3 kDa protein concentrator. Finally, SDS-PAGE analysis confirmed the acquisition of high-purity HRP2 protein with a molecular weight of approximately 9.2 kDa, which could be used for subsequent experiments. The amino acid sequence of HRP2 is shown in SEQ ID NO.2.

[0022] 2. Obtaining HiApt A 76nt nucleic acid sequence with high affinity for HRP2 was obtained through screening. The nucleotide sequence is shown in SEQ ID NO.1, specifically 5'-TTCAGCACTCCACGCATAGCTGTACCCGGCTGTACTGTCCGGGATGGTGTCTGAACCCTATGCGTGCTACCGTGAA-3'. This sequence was named HRP2-iAptamer, abbreviated as HiApt.

[0023] Predicting the secondary structure of HiApt using the Mfold online tool (e.g.) Figure 1 As shown in the figure, the results show that HiApt has a paired stem-ring structure.

[0024] Example 2: FP detection of the affinity between HiApt and HRP2 protein The experimental steps are as follows: 1. Experimental group preparation: The HiApt-FAM stock solution was diluted with MES buffer (pH 6.5) (30 mM MES, 25 mM NaCl, 0.002 mg / mL BSA, 2 mM β-mercaptoethanol, 1 mM CHAPS) to obtain a 100 nM HiApt-FAM solution. The HRP2 protein solution was serially diluted with pH 6.5 MES buffer to obtain 12 concentration gradients of HRP2 protein solutions (20 μM to 0.0095 μM). The 100 nM HiApt-FAM solution was mixed with equal volumes of protein solutions of 12 concentration gradients and incubated for 45 minutes to obtain experimental groups with a final HiApt-FAM concentration of 50 nM and HRP2 protein concentrations of 10, 5, 2.5, 1.25, 0.6250, 0.3125, 0.1562, 0.0780, 0.0390, 0.0195, 0.0095, and 0.0048 μM, respectively.

[0025] 2. Control group: HiApt-FAM solution diluted with pH 6.5 MES buffer to a final concentration of 50 nM.

[0026] 3. FP Detection: The experimental and control groups were subjected to fluorescence polarization scanning (spectral range 93.9-388 nM) using an EnVision 2105 microplate reader. Experimental data were processed and analyzed using a GraphPad Prism 9. Figure 2 As shown, the binding of HiApt to HRP2 protein conforms to the steady-state affinity model, with a KD value of 0.2912 ± 0.0074 μM.

[0027] Example 3: Flow cytometry detection of the affinity between HiApt and HRP2 protein 1. Protein coupling Dissolve the protein in ddH2O at a concentration of 1 mg / mL. Wash 50 μL of sugar beads (Cytiva product, catalog number: 17071601) three times with 100 μL of 1 mM glacial hydrochloric acid, centrifuge and discard the supernatant, then wash three times with 100 μL of DPBS, centrifuge and discard the supernatant. Add 50 μL of protein to the sugar beads and incubate at room temperature for 60 min. Wash twice with 100 μL of DPBS, centrifuge and discard the supernatant. Add twice the volume of the sugar beads of Buffer A (500 mM Methyllycine, 500 mM Sodium Chloride) and wash three times, then wash three times with Buffer B (100 mM Sodium Acetate Trihydrate, 500 mM Sodium Chloride). Wash three times with Buffer A, incubate at room temperature for 30 min, then wash three times with Buffer B, then wash three times with Buffer A, then wash three times with Buffer B again, centrifuge and discard the supernatant. Add twice the volume of BD sheathing solution and wash twice, centrifuge and discard the supernatant. Add 50 μL of BD sheathing solution and store at 4°C.

[0028] 2. Protein-nucleic acid incubation First, the HiApt-FAM gradient was diluted with DPBS, resulting in nine concentration gradients ranging from 200 nM to 0.78 nM. A blank sample was added to adjust the voltage. 10 μg of protein-bead was washed once with washing buffer, then resuspended in 1 mL of binding buffer. The bead was then incubated with different concentrations of nucleic acid for 30 min, washed twice with washing buffer, and then analyzed. Figure 3 As shown, the enhanced fluorescence intensity indicates that the protein has a good binding with HiApt-FAM. After systematic fitting, the binding of HiApt-FAM and HRP2 protein conforms to the steady-state affinity model, with a KD value of 9.4±1.8 nM.

[0029] WB: DPBS + 5mM Mg 2+ + 0.02% Tween BB: WB+1mg / mL BSA+0.1mg / mL HsDNA+5%FBS.

Claims

1. A nucleic acid aptamer that binds to the HRP2 protein, characterized in that, The nucleic acid aptamer includes a nucleotide sequence that has at least 70% homology with the nucleotide sequence shown in SEQ ID NO.1 and binds to the HRP2 protein; The nucleotide sequence shown in SEQ ID NO.1 is specifically TTCAGCACTCCACGCATAGCT GTACCCGGCTGTACTGTCCGGGATGGTGTCTGAACCCTATGCGTGCTACCGTGAA.

2. The nucleic acid aptamer for binding HRP2 protein according to claim 1, characterized in that, The nucleic acid aptamer includes the nucleotide sequence shown in SEQ ID NO.

1.

3. The nucleic acid aptamer for binding HRP2 protein according to claim 1 or 2, characterized in that, The nucleic acid aptamer comprises an RNA sequence transcribed from the nucleotide sequence shown in SEQ ID NO.

1.

4. A conjugate of the nucleic acid aptamer according to any one of claims 1-3, characterized in that, The conjugate is any substance used for labeling, detection, diagnosis, and treatment that is attached to the nucleotide sequence of the nucleic acid aptamer, and the conjugate of the nucleic acid aptamer after attaching the substance specifically binds to the HRP2 protein.

5. The conjugate according to claim 5, characterized in that, The substance includes at least one of the following: fluorescent marker, quencher group, azide group, alkynyl group, radioactive substance, therapeutic substance, biotin, digoxin, maleimide, nanomaterial, polyethylene glycol, small peptide, siRNA, and enzyme.

6. The use of any nucleic acid aptamer or derivative thereof according to any one of claims 1-3, or the conjugate according to claim 4 or 5, selected from any one of the following: 1) Purification or detection of HRP2 protein for non-diagnostic purposes; 2) Localization imaging of cells, tissues, or in vivo expressing HRP2 protein for non-diagnostic purposes; 3) As an inhibitor of HRP2 protein; 4) Prepare drugs that target the HRP2 protein.

7. The application according to claim 6, characterized in that, The amino acid sequence of the HRP2 protein is shown in SEQ ID NO.2; specifically: GSVEEKLQKLHSEIKFALKVGSPDVKRCLNALEELGTLQ VTSQILQKNTDVVATLKKIRRYKANKDVMEKAAEVYTRLKSR.

8. The use of any nucleic acid aptamer or derivative thereof according to any one of claims 1-3, or the conjugate according to claim 4 or 5, in the preparation of a medicament for targeting tumors.

9. The application according to claim 8, characterized in that, The tumors include liver cancer and / or colorectal cancer.

10. A drug targeting the HRP2 protein, characterized in that, It comprises at least one of the nucleic acid aptamers according to any one of claims 1-3, the conjugates according to claim 4 or 5, and derivatives of the nucleic acid aptamers according to any one of claims 1-3.

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