Nucleic acid aptamer combined with LDHA protein as well as application and screening method of nucleic acid aptamer

Abstract

The invention discloses a nucleic acid aptamer combined with LDHA protein, application and a screening method of the nucleic acid aptamer. The nucleic acid aptamer has a nucleotide sequence which has at least 60% homology with any one of SEQ ID NO.1 and SEQ ID NO.2 and is combined with LDHA protein; the nucleic acid aptamer which is small in molecular weight, stable in chemical property, easy to store and mark and capable of being combined with LDHA protein with high affinity and high specificity is obtained through screening by improving screening conditions through RNA sequences transcribed by nucleotide sequences shown in any one of SEQ ID NO.1 and SEQ ID NO.2, and the nucleic acid aptamer can be used for detecting the LDHA protein with high affinity and high specificity. The compound is relatively stable and simple in structure, easy to modify, capable of being artificially synthesized in a short time, stable in chemical property and easy to store and mark.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a nucleic acid aptamer binding to LDHA protein, and an application and screening method thereof. Background Art

[0002] Lactate dehydrogenase A chain (LDHA) consists of 332 amino acids and has 5 subtypes, which are mainly expressed in the cytoplasm. It exists in many tissues and organs of the human body, mainly distributed in skeletal muscle and liver. The main function of LDHA is to catalyze the conversion of acetone, NADH and H+ into lactic acid, NAD+ and H2O (the main function is to convert pyruvate into lactic acid during glycolysis), which is the last step of glycolysis.

[0003] LDHA plays a key role in the energy metabolism of tumors. Inhibiting LDHA activity can reduce cancer cell migration, invasion, angiogenesis and cancer metastasis. The reason is that in the absence of oxygen, tumor cells will also generate lactate and a small amount of ATP with the help of LDHA through glycolysis. This phenomenon is called the Warburg effect. The production of lactate will lead to a decrease in the pH value of the tumor cell microenvironment, which contributes to the growth and development of tumors. In addition, lactate can be transferred to the outside of the cell through specific transporters, affecting the surrounding tumor cells and promoting their proliferation and survival. Especially human metabolism-related cancers, including pancreatic cancer, laryngeal squamous cell carcinoma (LSCC), head and neck cancer, kidney cancer, gastric cancer, prostate cancer, breast cancer, hepatocellular carcinoma, oral squamous cell carcinoma (OSCC) and cervical cancer. At the same time, a large amount of literature data confirms that inhibiting LDHA activity will reduce tumor proliferation in various cancers such as head and neck cancer, pancreatic cancer, cervical cancer and prostate cancer.

[0004] In addition, LDHA is associated with resistance to radiotherapy for a variety of tumors. The main mechanism of killing cells by radiotherapy is to break the double-stranded DNA in the cell nucleus through the energy released by the rays, causing damage to the cell nucleus DNA. The NAD+ produced by LDHA can act as a substrate to participate in DNA repair through poly ADP ribose polymerase (PARP). In other words, only with the NAD+ provided by LDHA can PARP play a role in cell repair and resistance to death. At the same time, LDHA directly produces lactic acid, which causes the tumor inflammatory microenvironment to promote tumor radioresistance through immune escape mechanisms. Therefore, LDHA is a core factor in resistance to tumor radiotherapy.

[0005] In the field of scientific research, the study of LDHA usually involves its role as a biomarker and its application in disease diagnosis and treatment. However, at present, the specific mechanism of the LDHA axis in malignant tumors and related research on clinical diagnosis and treatment are not yet complete, and we still need to conduct more in-depth exploration and clinical verification, so as to improve the effects of targeted drugs and comprehensive treatments, and also help us find new predictive markers in malignant tumors to assist in early clinical diagnosis and postoperative prediction, and provide a basis for more accurate selection of LDHA targeted treatment targets, so as to achieve the purpose of individualized treatment and precision medicine, and bring new hope for malignant tumor patients to regain treatment.

[0006] Aptamers are DNA or RNA molecules separated by systematic evolution of ligands by exponential enrichment (SELEX). They can bind to other targets such as proteins, metal ions, small molecules, peptides, and even whole cells with high affinity and specificity, and therefore show broad prospects in biochemical analysis, environmental monitoring, basic medicine, and new drug synthesis. Compared with antibodies, aptamers have small molecular weight, better stability, easy modification, no immunogenicity, short production cycle, and can be synthesized artificially, eliminating a series of processes such as animal immunization, feeding, protein extraction, and purification. Therefore, if a nucleic acid aptamer with higher affinity and high specificity for LDHA protein can be found, it will help to achieve high-sensitivity and high-specificity detection of LDHA protein, and help develop drugs targeting LDHA protein.

[0007] Currently, there are no nucleic acid aptamers for LDHA protein. Therefore, it is necessary to find a nucleic acid aptamer that has high binding affinity for LDHA protein, good stability and specificity, is easy to modify and synthesize, and is easy to use. Summary of the invention

[0008] In order to overcome at least one of the defects described in the above-mentioned prior art, the present invention provides a nucleic acid aptamer binding to LDHA protein, its application and screening method, by screening, a nucleic acid aptamer with small molecular weight, stable chemical properties, easy storage and labeling, which can bind to LDHA protein with high affinity is obtained, which can be used for detection, diagnosis, imaging and treatment, and has broad application prospects.

[0009] The technical solution adopted by the present invention to solve the problem is:

[0010] A nucleic acid aptamer that binds to LDHA protein, the nucleic acid aptamer having a nucleotide sequence that is at least 60% homologous to any one of SEQ ID NO.1 and SEQ ID NO.2 and binds to LDHA protein; or an RNA sequence transcribed from the nucleotide sequence shown in any one of SEQ ID NO.1 and SEQ ID NO.2.

[0011] Furthermore, the nucleic acid aptamer has a nucleotide sequence as shown in any one of SEQ ID NO.1 and SEQ ID NO.2.

[0012] The present invention designs and synthesizes a random single-stranded DNA library and corresponding primers based on the SELEX technology, which are used to screen nucleic acid aptamers that have small molecular weight, stable chemical properties, are easy to store and label, and can bind to LDHA protein with high affinity. Thus, some nucleic acid aptamers that bind to LDHA protein with high affinity are screened, namely LDHA01 (SEQ ID NO.1) and LDHA05 (SEQ ID NO.2). The nucleic acid aptamers have higher affinity with LDHA protein and higher specificity.

[0013] For example, it may have an affinity for binding to LDHA protein that is equal to or higher than that of the parent nucleic acid aptamer sequence before modification, or it may have a higher stability although the affinity is not significantly improved. It is understood that the nucleotide sequence that has at least 30%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% homology with the nucleic acid aptamer provided by the present invention and binds to the LDHA protein, for example, the nucleotide sequence shown in any of the above nucleic acid aptamers may be deleted or a portion of the sequence may be added, and still have a high affinity for the LDHA protein, and still be within the scope of protection of the present invention.

[0014] In other words, the above aptamers, whether partially substituted or modified, have substantially the same or similar molecular structures, physicochemical properties and functions as the original aptamers, and can be used to bind to the LDHA protein.

[0015] Furthermore, the nucleotide sequence of the nucleic acid aptamer is modified, and the modification method includes one or more of phosphorylation, methylation, amination, sulfhydrylation, replacement of oxygen with sulfur, replacement of oxygen with selenium, and isotopization.

[0016] That is, a certain position on the nucleotide sequence of the above-mentioned nucleic acid aptamer can be modified, for example, phosphorylation, methylation, amination, sulfhydrylation, replacement of oxygen with sulfur, replacement of oxygen with selenium or isotopization, etc., provided that the nucleic acid aptamer sequence obtained after such modification has desirable properties, for example, it can have an affinity for binding to LDHA protein that is equal to or higher than that of the parent nucleic acid aptamer sequence before modification, or although the affinity is not significantly improved, it has higher stability.

[0017] Furthermore, the nucleic acid aptamer has a sequence that is at least 60% homologous to any one of SEQ ID NO.1 and SEQ ID NO.2 and is modified from a nucleotide sequence that binds to the LDHA protein.

[0018] Furthermore, the transformation includes at least one of the following transformation methods:

[0019] (1) Connecting a fluorescent marker to the aptamer;

[0020] (2) Connecting radioactive substances to the aptamer;

[0021] (3) Connecting therapeutic substances to the aptamer;

[0022] (4) connecting biotin to the aptamer;

[0023] (5) connecting digoxigenin to the aptamer;

[0024] (6) connecting a nanoluminescent material to the nucleic acid aptamer;

[0025] (7) Connecting a small peptide to the aptamer;

[0026] (8) Connecting siRNA to the nucleic acid aptamer.

[0027] By adopting the above scheme, the modified nucleic acid aptamer sequence has desirable properties, for example, it can have an affinity for binding to LDHA protein that is equal to or higher than that of the parent nucleic acid aptamer sequence before modification, or although the affinity is not significantly improved, it has higher stability.

[0028] The present invention also provides a derivative of a nucleic acid aptamer, which uses the above-mentioned nucleic acid aptamer that binds to the LDHA protein. The derivative of the nucleic acid aptamer includes a phosphorothioate skeleton or peptide nucleic acid that binds to the LDHA protein, which is modified from the nucleotide sequence skeleton of the nucleic acid aptamer or the conjugate of the nucleic acid aptamer.

[0029] By adopting the above scheme, the condition is that the derivatives all have molecular structures, physicochemical properties and functions that are substantially the same or similar to those of the original nucleic acid aptamer, and all bind to the LDHA protein.

[0030] The term "phosphorothioate backbone" as used in the present invention has the meaning generally understood by those of ordinary skill in the art, and refers to that the non-bridging oxygen atoms of the phosphodiester backbone of RNA and DNA aptamers can be replaced by one or two sulfur atoms, resulting in a phosphorothioate backbone with phosphorothioate or phosphorodithioate bonds, respectively. Such phosphorothioate backbones are known to have increased binding affinity to their targets, as well as enhanced resistance to nuclease degradation.

[0031] The term "peptide nucleic acid" used in the present invention has the meaning generally understood by those of ordinary skill in the art, and refers to an artificially synthesized DNA molecule analog, first reported by Nielsen et al. in 1991. Oligonucleotide mimics linked by peptide bonds were synthesized using N-2-(aminoethyl)-glycine units instead of sugar-phosphate backbones as repeating structural units, and are called peptide nucleic acids. Since peptide nucleic acids (PNA) do not have phosphate groups like those on DNA or RNA, there is a lack of electrical repulsion between PNA and DNA, making the binding strength between the two greater than the binding strength between DNA.

[0032] The present invention also provides a use of a nucleic acid aptamer and a nucleic acid aptamer derivative, specifically using the above-mentioned nucleic acid aptamer and nucleic acid aptamer derivative, and the use thereof includes at least one of the following:

[0033] (1) Quantitative or qualitative detection of LDHA protein;

[0034] (2) purifying LDHA protein;

[0035] (3) Imaging of LDHA protein;

[0036] (4) as an inhibitor of LDHA protein;

[0037] (5) preparing drugs targeting LDHA protein;

[0038] (6) preparing reagents or drugs for diagnosing and treating abnormal LDHA expression;

[0039] (7) Preparing inhibitors that block the binding of LDHA protein or LDHA protein to receptor protein.

[0040] In addition, the present invention also provides an LDHA protein detection product prepared using the nucleic acid aptamer. The product comprises the nucleic acid aptamer, or a conjugate or derivative of the nucleic acid aptamer.

[0041] Preferably, the product is a kit, which also includes a detection chip for use in conjunction with a chromatographic detection device.

[0042] The present invention also provides a method for screening a nucleic acid aptamer that binds to the LDHA protein, comprising the following steps:

[0043] S1, synthesize random single-stranded DNA library and primers;

[0044] S2. Magnetic bead screening: Perform at least 8 rounds of counter-screening and screening, and add serum in the 5th and 8th rounds of screening.

[0045] The serum is human serum (normal human serum, purchased from Beijing Solebow Technology Co., Ltd., item number: SL010).

[0046] Furthermore, in step S2, secondary screening (rounds 6-8) was introduced to reduce cross-contamination and PCR preference when screening many proteins with one library at the same time. At the same time, the specificity of the aptamer could be preliminarily determined based on the sequencing data. The specific process of adding serum in the 5th and 8th rounds of screening was as follows: 1% serum was added in the 5th round of screening, and 5% serum was added in the 8th round of screening.

[0047] By adopting the above scheme and adding serum during the magnetic bead screening process, the nucleic acid aptamer obtained by screening has higher affinity and better specificity, only binds to LDHA protein, does not bind to other proteins and can be used for the detection of LDHA protein.

[0048] The conventional conditions for nucleic acid aptamer screening are carried out in ion buffer, and a certain concentration of serum is gradually added under the screening conditions. On the one hand, because the serum contains rich proteins, it can compete with the proteins on the surface of the magnetic beads to bind to the library, thereby removing those sequences that have weak binding ability to the target LDHA protein or are only adsorbed. On the other hand, the aptamer binds to the target in the serum environment, which can better meet the actual detection environment of the later application based on nucleic acid aptamer development. At the same time, in the last few rounds of screening, nucleic acid aptamers with higher affinity and better specificity can be screened.

[0049] In summary, the present invention provides a method having the following technical effects:

[0050] 1. By improving the screening conditions, a nucleic acid aptamer with small molecular weight, stable chemical properties, easy storage and labeling, which can bind to LDHA protein with high affinity and high specificity, was screened;

[0051] 2. Nucleic acid aptamers have relatively stable and simple structures, are easy to modify, can be artificially synthesized in a short period of time, have stable chemical properties, and are easy to store and label;

[0052] 3. The nucleic acid aptamers obtained by the present invention can be used for LDHA protein detection, purification or imaging, and can also be used to prepare drugs for diagnosing or treating abnormal expression of LDHA protein, such as purification to obtain high-sensitivity and high-specificity detection of LDHA protein; can be used to prepare drugs targeting LDHA protein; can be used to prepare reagents or drugs for diagnosing or treating abnormal LDHA expression, etc., and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1Schematic diagram of the Cq value (A) and molecular retention rate (B) of the positive and negative screening elutions obtained by 8 rounds of screening using QPCR (real-time fluorescence quantitative PCR) in Example 1.

[0054] Figure 2 The coupling effect of LDHA protein and His was detected by flow cytometry in Example 1.

[0055] Figure 3 This is a graph showing the affinity detection data of nucleic acid aptamer LDHA01 and LDHA protein using SPR (surface plasmon resonance) in Example 2.

[0056] Figure 4 This is a graph showing the affinity detection data of nucleic acid aptamer LDHA05 and LDHA protein using SPR (surface plasmon resonance) in Example 2.

[0057] Figure 5 This is a graph showing the affinity detection data of nucleic acid aptamer LDHA01 and five other proteins using SPR (surface plasmon resonance) in Example 3.

[0058] Figure 6 This is a graph showing the affinity detection data of nucleic acid aptamer LDHA05 and five other proteins using SPR (surface plasmon resonance) in Example 3.

[0059] Figure 7 This is a schematic diagram of the results of the dot hybridization experiment based on the nucleic acid aptamer LDHA01 and the LDHA protein and control protein in Example 4.

[0060] Figure 8 This is a schematic diagram of the results of the dot hybridization assay based on the nucleic acid aptamer LDHA05 and the LDHA protein and control protein in Example 4.

[0061] Fig. 9 This is a schematic diagram of the results of the spot hybridization experiment based on the nucleic acid aptamer LDHA01 and different concentrations of LDHA protein in Example 5.

[0062] Fig.10 This is a schematic diagram of the results of the spot hybridization experiment based on the nucleic acid aptamer LDHA05 and different concentrations of LDHA protein in Example 5. DETAILED DESCRIPTION

[0063] For better understanding and implementation, the technical solutions in the embodiments of the present invention will be clearly and completely described and discussed below in conjunction with the accompanying drawings of the present invention. Obviously, what is described here is only a part of the examples of the present invention, not all the examples. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0064] In order to facilitate the understanding of the embodiments of the present invention, the following will be further explained by taking specific embodiments as examples in conjunction with the drawings, and each embodiment does not constitute a limitation on the embodiments of the present invention.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0066] The reagents not specifically mentioned in the present examples are all known products and were obtained by purchasing commercial products.

[0067] Example 1: Screening of ssDNA nucleic acid aptamers binding to LDHA protein

[0068] The method for screening ssDNA nucleic acid aptamers binding to LDHA protein in this embodiment comprises the following steps:

[0069] 1. Synthesize the random single-stranded DNA library and primers shown in the following sequence:

[0070] Based on SELEX technology, a random single-stranded DNA library, Lib10 library, was designed and synthesized:

[0071] 5'-ATTGGCACTCCACGCATAGG-N36-CCTATGCGTGCTACCGTGAA-3'; wherein "36N" represents a sequence formed by connecting 36 arbitrary bases. The library was synthesized by Shanghai Biotech Co., Ltd. (hereinafter referred to as "Shanghai Biotech") as the initial library.

[0072] 5'-ATTGGCACTCCACGCATAGG-N36-CCTATGCGTGCTACCGTGAA-3'; "36N" represents the sequence formed by connecting the top 1750 bases selected through sequencing results. This library was synthesized by Hangzhou Lianchuan Biotechnology Co., Ltd. (hereinafter referred to as "Lianchuan") as a secondary screening library.

[0073] Primer information is shown in Table 1 and was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0074] Table 1. Primer sequence information

[0075]

[0076]

[0077] The S in the primer name represents the forward primer, and the A represents the reverse primer.

[0078] The primers were prepared into 100 μM stock solution with ddH2O and stored at -40°C for future use.

[0079] 2. Magnetic bead screening

[0080] The magnetic bead method was used to screen LDHA protein nucleic acid aptamers, with a total of 8 rounds of screening. Rounds 1 to 5 were primary screening, and the initial library was synthesized by Bioengineering; rounds 6 to 8 were secondary screening, and the library was selected from rounds 1 to 5 of sequencing to select the top 1750 as the secondary screening library through Lianchuan synthesis. The purpose of secondary screening is to reduce cross-contamination and PCR preference when screening many proteins with one library at the same time, and at the same time, the specificity of the aptamer can be preliminarily judged based on the sequencing data. The specific screening pressure of each round is shown in Table 2.

[0081] Table 2. Screening pressure of LDHA protein aptamers

[0082]

[0083] DPBS buffer: potassium chloride 0.2 g / L, potassium dihydrogen phosphate 0.2 g / L, sodium chloride 8 g / L, disodium hydrogen phosphate dodecahydrate 2.8975 g / L, solvent is water, pH 7.4, stored at 25°C.

[0084] The specific screening process is as follows:

[0085] 1) Carboxyl magnetic beads coupled to LDHA protein (positive screening magnetic beads)

[0086] Take 50 μL of LDHA protein (purchased from ABclonal, Uniport ID: P00338, concentration is 1 mg / mL), add 550 μL of 10 mM sodium acetate buffer at pH 5.5 and mix well to make the protein positively charged, that is, the LDHA protein diluted with sodium acetate buffer, and place on ice for later use.

[0087] Take 300 μL of carboxyl magnetic beads (Jiangsu Zecheng Biotechnology Co., Ltd., catalog number: FM2221), wash once with 600 μL of 10mM NaOH aqueous solution, then wash twice with 600 μL of ultrapure water, fish the magnetic beads with a magnet, and discard the supernatant. Take 300 μL of the prepared NHS (N-hydroxysuccinimide; 0.1M aqueous solution) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution), mix equal volumes, add to the magnetic beads, and incubate at room temperature for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads. After the incubation, discard the supernatant and immediately wash once quickly with 600 μL of ultrapure water to reduce protein self-coupling. Add 600 μL of the LDHA protein diluted with the above sodium acetate buffer and incubate on a vertical mixer at room temperature for 60 minutes. The LDHA protein is coupled to the surface of the magnetic beads through the amino groups on the protein surface.

[0088] After the coupling is completed, place the coupling tube on a magnetic stand, discard the supernatant, take 600 μL of pH 8.5, 1M ethanolamine buffer and add it to the magnetic beads. Incubate on a vertical mixer at room temperature for 10 minutes to block the unreacted activation sites on the surface of the magnetic beads. Place on a magnetic stand and discard the blocking solution. 2+ After washing three times with DPBST buffer containing 0.02% Tween20, the beads were resuspended in 300 μL DPBS to obtain magnetic beads coupled with LDHA protein, marked as MB-LDHA, and stored at 4°C.

[0089] 2) Carboxyl magnetic beads coupled with His peptide (counter-screening magnetic beads) The same method as step 1), 600ul of carboxyl magnetic beads, LDHA protein replaced with 5μL His peptide (concentration of 20mg / mL), pH4.5 sodium acetate buffer, 10mM NaOH, ultrapure water, pH8.5 1M ethanolamine buffer, DPBST volume were changed to 1.2mL, resuspended DPBS was 0.6ml, other operations were the same, coupled with His peptide magnetic beads, labeled MB-His. His peptide is 9 consecutive histidines, synthesized by GenScript Biotech Co., Ltd.

[0090] 3) Detection of the coupling effect of LDHA protein and His, the specific operation is as follows:

[0091] Take 1ul anti-His-Aps + 100ul DPBS and mix well, mark as his antibody, and divide into 2 equal parts. One part is treated with 1ul LDHA protein-coupled magnetic beads + 50ul his antibody and mix well, marked as 53; the other part is treated with 1ul His-coupled magnetic beads + 50ul his antibody and mix well, marked as His. Incubate at room temperature in a vertical mixer for 1h in the dark. Place on a magnetic stand, discard the supernatant, retain the magnetic beads, wash 4 times with 100μL DPBST buffer, and resuspend with 600ul DPBS.

[0092] Prepare 1 μL of carboxyl magnetic beads, discard the supernatant by magnetic aspiration, wash 4 times with 100 μL of DPBST buffer, discard the supernatant by magnetic aspiration, add 600 μL of DPBS buffer and resuspend as a blank control, marked as Blank2.

[0093] Then the above three samples were detected by flow cytometry. The flow cytometry graph is shown in Figure 2 shown.

[0094] For blank magnetic beads, the flow fluorescence of the coupled LDHA protein and His both showed shift changes, indicating that both LDHA protein and His were attached to the magnetic beads.

[0095] 4) Library dissolution and denaturation treatment

[0096] The Lib10 random single-stranded nucleotide library (1OD) synthesized by Bioengineering was centrifuged at 12000rpm for 10 minutes, the library was centrifuged to the bottom of the tube, dissolved to 10μM with DPBS buffer, mixed and dispensed into PCR tubes for denaturation treatment. The treatment process is as follows: the PCR instrument is set to 95℃ for 10 minutes. The purpose of this step is to unwind the folded chain, then keep it at 4℃ for 5 minutes, and then equilibrate to room temperature for 2h. Then Mg2+ (final concentration of 5mM), BSA (final concentration of 0.05mg / mL), and HSDNA (final concentration of 0.05mg / mL) were added to the library and mixed to provide the ionic environment required for single-stranded DNA, reduce nonspecific binding and increase competitiveness. This is the library after denaturation treatment.

[0097] 5) Reverse screening

[0098] Take 50 μL MB-His, discard the supernatant by magnetic suction, wash twice with 200 μL DPBST buffer containing 5 mM Mg2+ and 0.02% Tween20, and add 100 μL of the library after renaturation treatment in step 4). After mixing, incubate at room temperature for 60 minutes on a vertical mixer. Place it on a magnetic stand, collect the supernatant, mark it as Pool-1, and use the supernatant as a single-stranded nucleic acid library for positive screening with MB-LDHA magnetic beads. Wash the remaining magnetic beads 4 times with 200 μL DPBS buffer. Finally, add 100 μL ultrapure water to the washed magnetic beads, boil them in a boiling water bath for 10 minutes, collect the supernatant (i.e., the reverse screening supernatant), and mark it as elution-His.

[0099] 6) Positive screening

[0100] Take 50 μL MB-LDHA, discard the supernatant by magnetic aspiration, and wash with 200 μL containing 5 mM Mg 2+ , 0.02% Tween20 DPBST buffer solution, wash twice, discard the supernatant, add 100 μL of the anti-screening supernatant Pool-1 collected in step 5) and mix with it, and incubate at room temperature for 60 minutes on a vertical mixer. Place on a magnetic stand, aspirate and discard the supernatant, retain the magnetic beads, and wash the magnetic beads 4 times with 200 μL DPBS buffer. Finally, add 100 μL of ultrapure water to the washed magnetic beads, boil in a boiling water bath for 10 minutes, collect the supernatant, and mark it as elution-LDHA.

[0101] 7) Preparation of secondary library

[0102] 10 μL step 6) elution-LDHA and elution-His in the reverse screening process were quantified by QPCR, and the screening process was monitored by calculating the number of molecules retained in each round. The results are shown in Figure 1 -B.

[0103] The nucleic acid molecules in step 6) elution-LDHA were used as templates and amplified by ordinary PCR. The method is as follows: add all template elution-LDHA to 1.8ml PCR mix and mix well, divide the template and PCR mix mixture into 100μL / tube and add it to the PCR tube, and the amplification conditions are as follows: 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 30 seconds, a total of 25 cycles, 72℃ total extension for 3 minutes, and storage at 4℃. The PCR raw materials in the PCR mix are purchased from Novozymes' dNTPMix (P031-02-AA) and TaKaRa's Taq enzyme (R001WZ).

[0104] The amplified product was purified using commercial Tiandirenhe SA magnetic beads (SM017010) purchased to prepare the secondary library for the next round of screening. Prepare 80μLSA magnetic beads, wash twice with 500μL DPBST buffer containing 5mM Mg2+ and 0.02% Tween20, discard the supernatant by magnetic suction, add 2ml of the PCR product in step 5), and then add 1 / 5 volume of 4M sodium chloride aqueous solution of the PCR product, incubate on a shaker at room temperature for 30 minutes, and discard the supernatant by magnetic suction. Use 500μL DPBST (containing 5mM Mg2+, 0.02% Tween20) buffer to wash the magnetic beads three times, discard the supernatant, add 100μL of 40mM sodium hydroxide aqueous solution, incubate at room temperature for three minutes, and then remove the magnetic beads by magnetic suction. Then add 4 μL of 1M hydrochloric acid to the supernatant to neutralize the single chain, and then add 104 μL of 2*DPBS buffer to dilute and neutralize the salt concentration, and finally obtain 208 μL of the secondary library dissolved in 1*DPBS buffer, which can be used as the library for the next round of screening, that is, the first round of screening is completed.

[0105] The magnetic bead method was repeated for 8 rounds of screening. From the 2nd to the 8th round, the secondary library obtained in the previous operation was used as the starting nucleic acid library for each operation, and the operations were performed according to steps 4), 5), 6) and 7).

[0106] 8) Library affinity detection

[0107] The enrichment of the library can be determined based on the QPCR Cq value of elution during each round of positive and negative screening. If the number of templates eluted from each round of positive screening increases (Cq advances), Figure 1 -A. Figure 1 -A The retention rate of each round of molecules was calculated based on the elution Cq values ​​of the positive and negative screens. The retention rate of the positive screen gradually increased, indicating that the library was effectively enriched. Figure 1 -B.

[0108] 3. Analysis and identification of nucleic acid aptamers obtained after screening: After high-throughput sequencing analysis of the obtained enriched library products, 12 sequences were selected and synthesized by GENEWIZ Biotechnology (Jiangsu) Technology Co., Ltd., and the affinity was detected using the gold standard surface plasmon resonance (SPR).

[0109] In subsequent tests, two sequences with the strongest binding ability were identified from the sequences obtained in the final 8th round. They were nucleic acid aptamers with nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2. They were named LDHA01 and LDHA05, respectively. The specific sequences are shown in Table 3.

[0110] Table 3. LDHA01 and LDHA05 sequence listing

[0111]

[0112] Example 2: Surface Plasmon Resonance (SPR) Detection of Affinity between LDHA Protein Aptamer and LDHA Protein

[0113] Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize nucleic acid aptamers LDHA01 (SEQ ID NO.1) and LDHA05 (SEQ ID NO.2), which were respectively washed with DPBS buffer (containing 5 mM Mg 2+ ) were diluted to 500nM, 250nM, 125nM, 62.5nM, and 31.25nM.

[0114] 1. The LDHA protein was coupled to the 2nd channel of the 7th channel on the surface of the CM5 chip of the surface plasmon resonance instrument (GE Healthcare, model: Biacore8K) as the experimental channel. The specific method is as follows: Equal volumes of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1M aqueous solution) were mixed and injected into the activation chip at a flow rate of 10 μL / min. The LDHA protein (purchased from ABclonal, Uniport ID: P00338, concentration of 1 mg / mL) was diluted with 10 mM sodium acetate buffer at pH 5.5 to a final concentration of 50 μg / mL and then injected. The injection volume was 190 μL, the flow rate was 30 μL / min, and the LDHA protein coupling amount was about 13110.8 RU. After the injection was completed, the chip was sealed with ethanolamine, the flow rate was 30 μL / min, and 129 μL was injected.

[0115] The operation was the same as that of the first channel of the 7th channel, except that the LDHA protein was replaced by the His protein (same as in Example 1). Other operations were the same, and the His protein was coupled to the first channel of the 7th channel as a control channel.

[0116] 2. Detection: Use DPBS containing 5mM Mg2+ to dilute the nucleic acid aptamer LDHA01 to 500nM, 250nM, 125nM, 62.5nM, and 31.25nM. Then flow through the 1st and 2nd channels of the 7th channel respectively. The procedure of each channel is as follows: the nucleic acid aptamer is injected at 30μL / min for 2 minutes; DPBS buffer (containing 5mM Mg2+) 2+ ) was injected at 30 μL / min for dissociation, the time was 2 minutes; 1.5M NaCl aqueous solution was injected at 30 μL / min for regeneration, the time was 30 seconds. Figure 3 shown.

[0117] Under the same conditions, the nucleic acid aptamer LDHA01 was replaced by the nucleic acid aptamer LDHA05, and other operations were the same. The results are shown in Figure 4 shown.

[0118] The affinity KD values ​​of LDHA protein nucleic acid aptamers LDHA01 and LDHA05 with LDHA protein are shown in Table 4.

[0119] Table 4. Affinity of nucleic acid aptamers LDHA01, LDHA05 and LDHA protein

[0120] Aptamers length Affinity KD (nM) LDHA01 (SEQ ID NO. 1) 76 31.7 LDHA05 (SEQ ID NO. 2) 76 4.5

[0121] Example 3: Study on the specificity of nucleic acid aptamers

[0122] TIM-3 protein (catalog number: 10390-H08H, purchased from Sino Biological), CD79B protein (catalog number: RP01464, purchased from ABclonal), PTK7 protein (catalog number: P02293QB, purchased from Younuozhen Biotechnology), FAP protein (catalog number: C14G, purchased from Jinan Protein), Nectin-4 protein (catalog number: CJ19, purchased from Jinan Protein).

[0123] In this embodiment, TIM-3 protein, CD79B protein, PTK7 protein, FAP protein, and Nectin-4 protein are used instead of LDHA protein, respectively. The method of fixing LDHA protein to the SPR chip for testing is the same as that in Example 2. TIM-3 protein, CD79B protein, PTK7 protein, FAP protein, and Nectin-4 protein are coupled to the second channel of the six channels on the surface of the CM5 chip, respectively, and the coupling amounts are 1597.7U, 3920.3RU, 7483.4RU, 9768.0RU, and 3666.9RU, respectively. The first channels of the six channels are all coupled with His, and two nucleic acid aptamers, LDHA01 and LDHA05, diluted to 500nM, are injected sequentially.

[0124] The affinity test data of nucleic acid aptamer LDHA01 and the above five proteins are shown in Figure 5 , the affinity test data of nucleic acid aptamer LDHA05 and the above five proteins are shown in Figure 6 .Depend on Figure 5 .6 It can be seen that the nucleic acid aptamers LDHA01 and LDHA05 cannot bind to the five proteins TIM-3, CD79B, PTK7, FAP, and Nectin-4. It can be seen that the nucleic acid aptamer of LDHA that has been screened and obtained has a very high specificity in binding to the target.

[0125] Example 4: Detection of LDHA protein by dot blot hybridization assay based on nucleic acid aptamers

[0126] Suzhou Genewiz Biotech Co., Ltd. was commissioned to synthesize biotin-modified nucleic acid aptamers LDHA01 and LDHA05.

[0127] Dot hybridization experiments were performed on the biotin-modified nucleic acid aptamers LDHA01 and LDHA05, respectively, and the steps were as follows:

[0128] 1. Detection of specificity of LDHA protein by dot blot hybridization assay with nucleic acid aptamers LDHA01 and LDHA05

[0129] (1) LDHA protein was dissolved in DPBS buffer to 0.500 mg / mL as the test sample; control proteins His peptide, BSA, EPCAM, Fibrinogen, and holo-Transferrin were dissolved in DPBS buffer to 0.500 mg / mL as the control sample. Take a 8 cm × 2 cm test strip (nitrocellulose membrane, 0.2 μm, GE Amersham TM , Catalog No. 10600004), spot 2 μL of the test sample and control sample respectively onto the nitrocellulose membrane and air-dry for 40 minutes.

[0130] (2) Then place the test strip in a 50 mL centrifuge tube, add 6 mL of DPBS buffer containing 10% BSA to soak it, and block it on a shaker at room temperature for 1 hour. After blocking, use DPBST (containing 5 mM Mg 2+ , 0.02% Tween20) for 5 min, repeat the washing for 3 times and aspirate clean.

[0131] (3) Transfer the test strip to a new 50 mL centrifuge tube and add 6 mL of DPBS buffer (containing 5 mM Mg 2+ ) dissolved in 500 nM biotin-modified aptamer LDHA01, incubated on a shaker at room temperature for 30 min, and then washed with DPBST (containing 5 mM Mg 2+ , 0.02% Tween20) for 2 min, repeat the washing for 3 times and aspirate clean.

[0132] (4) Transfer the test strip to a new 50 mL centrifuge tube, add 6 mL of HRP-Streptavidin (1 mg / ml purchased from Beyotime Biotech): DPBS buffer = 1:2000 dilution to soak it, and incubate it on a shaker at room temperature for 30 min. After the incubation, use DPBST (containing 5 mM Mg 2+ , 0.02% Tween20) for 2 min, repeat the washing for 3 times and aspirate clean.

[0133] (5) Transfer the test strip to a clean PE glove, take an ECL color development kit (purchased from Beyotime Biotech), mix 100 μL of solution A and 100 μL of solution B, soak the surface of the test strip, and incubate in the dark for 5 min.

[0134] (6) Observation and photography by imaging system: Amersham ImageQuant 800 digital imaging system was used for photography. The results are shown in Figure 7 .

[0135] Under the same conditions, 500 nM biotin-modified aptamer LDHA01 was replaced with 500 nM biotin-modified aptamer LDHA05. The results are shown in Figure 8 .

[0136] Depend on Figure 7 and Figure 8 It can be seen that compared with the spots of the control proteins His peptide, BSA, EPCAM, Fibrinogen, and holo-Transferrin, the LDHA protein is obvious. This shows that the biotin-modified nucleic acid aptamers LDHA01 and LDHA05 can be used for the detection of membrane hybridization LDHA protein, and the control proteins His peptide, BSA, EPCAM, Fibrinogen, and holo-Transferrin are almost colorless, which further proves that LDHA01 and LDHA05 can specifically bind to the LDHA protein.

[0137] 2. Detection of LDHA protein at different concentrations by dot blot hybridization assay with nucleic acid aptamers LDHA01 and LDHA05

[0138] Dissolve LDHA protein in DPBS buffer to 0.500 mg / mL, 0.250 mg / mL, 0.125 mg / mL, 0.063 mg / mL, 0.032 mg / mL, and 0.016 mg / mL, and spot 4 μL of each onto the nitrocellulose membrane. Other operations are the same as step 1.

[0139] The results of the dot hybridization experiment of 500nM biotin-modified aptamer LDHA01 and different concentrations of LDHA protein are shown in Fig. 9 As shown; the results of the spot hybridization experiment of 500nM biotin-modified nucleic acid aptamer LDHA05 and different concentrations of LDHA protein are shown Fig.10 shown.

[0140] Depend on Fig. 9 and Fig.10 It can be demonstrated that two of the nucleic acid aptamers can be used to accurately detect 0.125 mg / ml of LDHA protein.

[0141] The technical means disclosed in the scheme of the present invention are not limited to the technical means disclosed in the above-mentioned implementation mode, but also include technical schemes composed of any combination of the above-mentioned technical features. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also regarded as the protection scope of the present invention.

Claims

1. A nucleic acid aptamer binding to LDHA protein, characterized in that: The nucleic acid aptamer has a nucleotide sequence that is at least 60% homologous to any one of SEQ ID NO.1 and SEQ ID NO.2 and binds to the LDHA protein; or an RNA sequence transcribed from the nucleotide sequence shown in any one of SEQ ID NO.1 and SEQ ID NO.

2.

2. The nucleic acid aptamer binding to LDHA protein according to claim 1, characterized in that: The nucleic acid aptamer has a nucleotide sequence as shown in any one of SEQ ID NO.1 and SEQ ID NO.

2.

3. The nucleic acid aptamer binding to LDHA protein according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid aptamer is modified, and the modification method includes one or more of phosphorylation, methylation, amination, sulfhydrylation, replacement of oxygen with sulfur, replacement of oxygen with selenium, and isotopization.

4. The nucleic acid aptamer binding to LDHA protein according to claim 2, characterized in that: The nucleic acid aptamer has a sequence that is at least 60% homologous to any one of SEQ ID NO.1 and SEQ ID NO.2 and is obtained by modifying the nucleotide sequence that binds to the LDHA protein.

5. The nucleic acid aptamer binding to LDHA protein according to claim 2, characterized in that: The transformation includes at least one of the following transformation methods: (1) Connecting a fluorescent marker to the aptamer; (2) Connecting radioactive substances to the aptamer; (3) Connecting therapeutic substances to the aptamer; (4) connecting biotin to the aptamer; (5) connecting digoxigenin to the aptamer; (6) connecting a nanoluminescent material to the nucleic acid aptamer; (7) Connecting a small peptide to the aptamer; (8) Connecting siRNA to the nucleic acid aptamer.

6. A derivative of a nucleic acid aptamer, comprising the nucleic acid aptamer for binding to LDHA protein according to any one of claims 1 to 5, characterized in that: The derivatives of the nucleic acid aptamer include a phosphorothioate backbone or a peptide nucleic acid that binds to the LDHA protein and is formed by modifying the nucleotide sequence backbone of the nucleic acid aptamer or the conjugate of the nucleic acid aptamer.

7. The use of the nucleic acid aptamer according to any one of claims 1 to 6 and the nucleic acid aptamer derivative according to claim 8, characterized in that: Include at least one of the following: (1) Quantitative or qualitative detection of LDHA protein; (2) purifying LDHA protein; (3) Imaging of LDHA protein; (4) as an inhibitor of LDHA protein; (5) preparing drugs targeting LDHA protein; (6) preparing reagents or drugs for diagnosing and treating abnormal LDHA expression; (7) Preparing inhibitors that block the binding of LDHA protein or LDHA protein to receptor protein.

8. A method for screening nucleic acid aptamers binding to LDHA protein, characterized in that: The following steps are involved: S1, synthesize random single-stranded DNA library and primers; S2. Magnetic bead screening: Perform at least 8 rounds of counter-screening and screening, and add serum in the 5th and 8th rounds of screening.

9. The method for screening a nucleic acid aptamer binding to LDHA protein according to claim 8, characterized in that: In step S2, secondary screening (rounds 6-8) was introduced to reduce cross-contamination and PCR preference when screening many proteins with one library at the same time. At the same time, the specificity of the aptamer could be preliminarily determined based on the sequencing data. The specific process of adding serum in the 5th and 8th rounds of screening was as follows: 1% serum was added in the 5th round of screening, and 5% serum was added in the 8th round of screening.

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