Nucleic acid aptamer combined with lung cancer serum, target identification and application thereof

Abstract

The invention discloses a nucleic acid aptamer combined with lung cancer serum, target identification and application of the nucleic acid aptamer. The nucleic acid aptamer is obtained by screening the lung cancer serum through a paramagnetic particle method, and the sequence of the nucleic acid aptamer is shown as SEQ ID NO.1-SEQ ID NO.10. According to the nucleic acid aptamer with the optimal affinity, the target protein TGFBI specifically recognized in lung cancer serum is called through a streptavidin-biotin method, it is detected that SEQ ID NO.3 and the TGFBI protein have good affinity and specificity, and the potential of the TGFBI protein as a lung cancer tumor marker is shown; the feasibility of the aptamer for screening tumor markers is verified, and a new thought is provided for early screening and targeted therapy of cancers.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a nucleic acid aptamer combined with lung cancer serum, target identification and application thereof. Background Art

[0002] Lung cancer has been one of the most common cancers in men and women worldwide for the past few decades. Despite advances in diagnosis and treatment in recent years, lung cancer still accounts for a high proportion of cancer-related deaths and has a low 5-year survival rate. Lung cancer is characterized by uncontrolled proliferation of lung tissue cells. Uncontrolled proliferation without timely treatment will quickly spread to tissues near the lungs and eventually to other parts of the body. Lung cancer is divided into central and peripheral types according to location. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) according to its biological characteristics, of which non-small cell lung cancer accounts for about 80%. NSCLC includes large cell carcinoma, adenocarcinoma, squamous cell carcinoma and other types. The mortality rate of lung cancer is very high, and it is difficult to make an accurate and effective early diagnosis. Most patients are diagnosed with advanced lung cancer. Early diagnosis and treatment are crucial to improving patient survival and quality of life. At present, diagnostic methods for lung cancer, such as imaging examinations and histopathological analysis, have certain limitations and shortcomings, so finding new, more sensitive and specific lung cancer tumor markers has become an important research direction.

[0003] Aptamers are artificial oligonucleotides (DNA or RNA) composed of dozens of nucleotides. They can bind to different types of targets with high affinity and specificity, such as peptides, proteins, drugs, organic and inorganic molecules, and even whole cells. Aptamers are produced by an in vitro selection process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX), which is screened from a synthetic oligonucleotide library, including iterative cycles of binding, separation, and amplification. The high affinity and specificity of aptamers depend on their ability to adaptively fold through pairing between certain complementary bases within the chain, as well as electrostatic interactions, hydrogen bonding, van der Waals forces, and base stacking, forming some stable three-dimensional spatial structures, such as pockets, hairpins, pseudoknots, G-tetramers, etc. In theory, they can recognize and bind to all types of target molecules through these special structures. Nucleic acid aptamers are also called "chemical antibodies". Compared with monoclonal antibodies, nucleic acid aptamers have no immunogenicity and have unique advantages such as a wide target range, small molecular weight, low toxicity, easy in vitro chemical synthesis and modification and labeling, and low transportation and storage costs. Therefore, since the concept of nucleic acid aptamers was proposed, it has received widespread attention from the scientific research community.

[0004] Transforming Growth Factor Beta Induced Protein (TGFBI) is an extracellular matrix protein induced by transforming growth factor (TGF)-β1 and has multiple biological functions. The current detection of serum tumor markers for lung cancer still has some limitations, for example, the false positive rate of markers such as CEA and CYFRA21-1 is high. Some lung cancer tumor markers are not ideal in sensitivity and specificity in early diagnosis. Therefore, screening for serum tumor markers with high sensitivity and specificity for lung cancer is still the main trend in early diagnosis of lung cancer. Therefore, using nucleic acid aptamers as probes and utilizing their high specificity and high affinity interactions with target proteins, more effective and sensitive lung cancer tumor markers can be obtained, thereby promoting early detection and diagnosis of lung cancer. Summary of the invention

[0005] 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 that binds to lung cancer serum, and successfully identifies its specifically bound target protein in lung cancer serum, thereby providing a new tumor marker for the early diagnosis of lung cancer, and further providing a new approach for the early detection and treatment of lung cancer.

[0006] The technical solution adopted by the present invention to solve the problem is:

[0007] Based on the SELEX technology, a random ssDNA library and corresponding primers were designed and synthesized. By screening lung cancer serum, a nucleic acid aptamer with small molecular weight, stable chemical properties, easy storage and labeling, which can bind to lung cancer serum with high affinity, was obtained as shown in SEQ ID NO.1 to SEQ ID NO.10, and the target protein TGFBI with specific binding was successfully identified in lung cancer serum using the optimal binding nucleic acid aptamer. The amino acid sequence of the protein is shown in SEQ ID NO.11.

[0008] A nucleic acid aptamer that binds to lung cancer serum, characterized in that the nucleic acid aptamer has a nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO.10.

[0009] Furthermore, a nucleic acid aptamer that binds to TGFBI protein has a nucleotide sequence that is at least 60% homologous to SEQ ID NO.3 and binds to TGFBI protein.

[0010] 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 TGFBI protein, for example, the nucleotide sequence shown in the above nucleic acid aptamer can be deleted or added, and still has high affinity with the TGFBI protein, and is still within the scope of protection of the present invention. Further, the nucleic acid aptamer has an RNA sequence transcribed from the nucleotide sequence shown in SEQ ID NO.3.

[0011] Furthermore, the nucleic acid aptamer has a sequence obtained by truncating the nucleotide sequence shown in SEQ ID NO.3.

[0012] Furthermore, the nucleic acid aptamer includes a sequence obtained by modifying a nucleotide sequence that has at least 60% homology to SEQ ID NO. 3 and binds to the TGFBI protein.

[0013] Furthermore, the modification includes at least one of the following modification methods:

[0014] (1) Phosphorylation;

[0015] (2) Methylation;

[0016] (3) Amination;

[0017] (4) thiolation;

[0018] (5) Isotopization;

[0019] (6) Substituting sulfur for oxygen;

[0020] (7) Replace oxygen with selenium.

[0021] In some ways, as an improvement on the above technical solution, 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 TGFBI 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.

[0022] Therefore, in some embodiments, the nucleotide sequence of the nucleic acid aptamer is modified and the modified nucleic acid aptamer specifically binds to the TGFBI protein, and the modification is selected from at least one of phosphorylation, methylation, amination, thiolation, replacement of oxygen with sulfur, replacement of oxygen with selenium and isotopization, which is still within the scope of protection of the present invention.

[0023] Furthermore, the nucleic acid aptamer has a sequence obtained by modifying a nucleotide sequence that has at least 60% homology to SEQ ID NO.3 and binds to the TGFBI protein;

[0024] The transformation includes at least one of the following transformation methods:

[0025] (1) Connecting a fluorescent marker to the aptamer;

[0026] (2) Connecting radioactive substances to the aptamer;

[0027] (3) Connecting therapeutic substances to the aptamer;

[0028] (4) connecting biotin to the nucleic acid aptamer;

[0029] (5) connecting digoxigenin to the aptamer;

[0030] (6) connecting a nanoluminescent material to the nucleic acid aptamer;

[0031] (7) Connecting a small peptide to the nucleic acid aptamer;

[0032] (8) Connecting siRNA to the nucleic acid aptamer.

[0033] The nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO.3; the conjugate of the nucleic acid aptamer includes a fluorescent marker, etc.; the derivative of the nucleic acid aptamer includes a thiophosphate skeleton or peptide nucleic acid that binds to the TGFBI protein, which is modified from the nucleotide sequence skeleton of the nucleic acid aptamer or the conjugate of the nucleic acid aptamer.

[0034] The conjugate of nucleic acid aptamer described in the present invention refers to connecting other groups to the nucleic acid aptamer, such as fluorescent markers with marking functions, such as FAM, radioactive substances, therapeutic substances, biotin, digoxin, nanoluminescent materials, small peptides, siRNA or enzyme labels, etc., so that the nucleic acid aptamer sequence obtained after modification has desirable properties, for example, it can have an affinity for binding to TGFBI 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.

[0035] 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 TGFBI protein.

[0036] In addition, the present invention also provides nucleic acid aptamer derivatives, which are obtained by transforming the nucleotide sequence skeleton of the above-mentioned nucleic acid aptamer into a phosphorothioate skeleton that binds to the TGFBI protein, or are peptide nucleic acids that bind to the TGFBI protein that are transformed from the nucleic acid aptamer or the conjugate of the nucleic acid aptamer in any of the above-mentioned technical solutions. The condition is that the derivatives have a molecular structure, physicochemical properties and functions that are basically the same or similar to the original nucleic acid aptamer, and all bind to the TGFBI protein.

[0037] 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.

[0038] 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.

[0039] The present invention also provides a use of the above-mentioned nucleic acid aptamer and the above-mentioned nucleic acid aptamer derivative, including at least one of the following:

[0040] (1) Detection of TGFBI protein;

[0041] (2) localization imaging of cells, tissues or living organisms expressing TGFBI;

[0042] (3) capturing cells or exosomes expressing TGFBI;

[0043] (4) Drug conjugation;

[0044] (5) Targeted delivery of drugs to tumor cells;

[0045] (6) Regulate TGFBI protein function;

[0046] (7) preparing TGFBI protein detection reagent;

[0047] (8) preparing imaging agents;

[0048] (9) preparing drug carriers;

[0049] (10) Preparation of anti-tumor drugs.

[0050] The present invention also provides a screening method based on lung cancer serum nucleic acid aptamers, comprising the following steps:

[0051] S1, synthesizing single-stranded ssDNA library and primers;

[0052] S2, magnetic bead screening: lung cancer sera were screened for 7 rounds, and counter-screening was added in rounds 8-11. The conventional conditions for screening were carried out in ionic buffer;

[0053] S3, high-throughput sequencing, selecting multiple aptamers with high abundance rankings and from different families;

[0054] S4. Using real-time fluorescence quantitative PCR (qPCR) to verify the binding ability of multiple nucleic acid aptamers, and obtain the nucleic acid aptamer that best binds to lung cancer serum;

[0055] S5. The optimal binding nucleic acid aptamer is used to identify the specific binding target in lung cancer serum through the streptavidin-biotin method.

[0056] The beneficial effects of the nucleic acid aptamer binding to TGFBI protein in lung cancer serum provided by the present invention are:

[0057] 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 lung cancer serum with high affinity, was screened out;

[0058] 2. The specific binding protein TGFBI of the nucleic acid aptamer and lung cancer serum was identified;

[0059] 3. The nucleic acid aptamer has a relatively stable and simple structure, is easy to modify, can be artificially synthesized in a short period of time, has stable chemical properties, and is easy to store and label;

[0060] 4. The nucleic acid aptamers obtained by the present invention can be used in TGFBI protein detection, diagnosis, imaging and treatment, such as purification or high-sensitivity detection of TGFBI protein; preparation of drugs targeting TGFBI protein; reagents or drugs for diagnosis and treatment of abnormal TGFBI protein expression, etc., and have broad application prospects.

[0061] 5. The present invention adopts an improved screening method, which effectively improves the specificity of the screened nucleic acid aptamers by screening with magnetic beads combined with a counter-screening strategy. This method can reduce the binding with non-specific substances, thereby reducing the probability of false positives.

[0062] 6. The present invention clearly points out that the screened nucleic acid aptamer specifically binds to the TGFBI protein, which is very important for subsequent application development because a clear target helps to diagnose and treat diseases more accurately. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a schematic diagram of the molecular retention rates obtained in the 1st, 4th, 7th, 8th, 9th, 10th and 11th rounds of screening using qPCR (real-time fluorescence quantitative PCR) to detect the positive and negative elution Cq values ​​obtained in 11 rounds of screening in Example 1.

[0064] Figure 2 This is a schematic diagram of the binding ability of the enriched library obtained in the 1st, 5th, 9th and 11th rounds of screening detected by flow cytometry in Example 1 with the counter-screening control HSA human serum albumin and the positive screening target lung cancer serum.

[0065] Figure 3 The Cq value of the binding of the nucleic acid aptamer to the positive screening target and the reverse screening target verified by qPCR in Example 2.

[0066] Figure 4 It is the concentration ratio calculated by using qPCR to detect the binding Cq value of nucleic acid aptamer DH03 with the positive screening target and the reverse screening target in Example 2.

[0067] Figure 5 This is a schematic diagram of the affinity test results between nucleic acid aptamer DH03 and TGFBI protein in Example 4.

[0068] Figure 6 This is a schematic diagram of the specificity study results of nucleic acid aptamer DH03 and TGFBI protein in Example 5.

[0069] Figure 7 Schematic diagram of the results of the dot hybridization assay of nucleic acid aptamer DH03 and different concentrations of TGFBI protein in Example 6. DETAILED DESCRIPTION

[0070] 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.

[0071] 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.

[0072] 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.

[0073] The present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto:

[0074] The reagents not specifically mentioned in the present examples are all known products and were obtained by purchasing commercial products.

[0075] Example 1: Screening of ssDNA aptamers binding to lung cancer serum

[0076] The method for screening nucleic acid aptamers binding to lung cancer serum ssDNA in this embodiment comprises the following steps:

[0077] 1. Synthesize the random ssDNA library and primers shown in the following sequences:

[0078] Based on SELEX technology, a random ssDNA library, Library10, was designed and synthesized:

[0079] 5'-ATTGGCACTCCACGCATAGG-N36-CCTATGCGTGCTACCGTGA A-3' (N=A, G, T, C). This library was synthesized by Shanghai Biotech Co., Ltd. (hereinafter referred to as "Shanghai Biotech").

[0080] Primer information is shown in Table 1 and was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0081] Table 1. Primers and their sequences

[0082]

[0083] The S in the primer name represents the forward primer, and the A in the primer name represents the reverse primer. The primers were prepared into 100 μM storage solutions with ultrapure water and stored at -20°C for future use.

[0084] 2. Magnetic bead screening

[0085] The magnetic bead method was used to screen lung cancer serum aptamers, and a total of 11 rounds of screening were performed. The screening pressure adjustment for each round is shown in Table 2.

[0086] Table 2. Screening pressure of lung cancer serum aptamers

[0087]

[0088] 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 ultrapure water, pH 7.4, stored at 25°C.

[0089] The specific screening process is as follows:

[0090] 1) Carboxyl magnetic beads (positive screening magnetic beads) coupled to homogenized lung cancer serum

[0091] In this experiment, Protein Enrichment Kits (Bio-Rad) were used to process mixed lung cancer serum (lung adenocarcinoma samples provided by the Laboratory Department of Zhejiang Cancer Hospital) to remove high-abundance proteins in the serum and enrich low-abundance proteins, thereby achieving the purpose of homogenization and coupling all proteins to the carboxyl magnetic beads as much as possible to improve the success rate of screening.

[0092] Take 500 μL of carboxyl magnetic beads (Jiangsu Zecheng Biotechnology Co., Ltd., catalog number: FM2221), wash once with 500 μL of 20mM NaOH aqueous solution, then wash twice with 500 μL of ultrapure water, fish the magnetic beads with a magnet, and remove the supernatant. Take 500 μL of the prepared NHS (N-hydroxysuccinimide; 0.1M aqueous solution) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution), mix them in equal volumes, add them to the magnetic beads, and incubate at 25°C for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads. After the incubation, remove the supernatant and immediately wash once quickly with 500 μL of ultrapure water to reduce protein self-coupling. Add 125 μg of the lung cancer serum after the above homogenization treatment, incubate on a vertical mixer at 25°C for 60 minutes, and the protein in the lung cancer serum is coupled to the surface of the magnetic beads through the amino groups on the protein surface.

[0093] After the coupling is completed, the supernatant is discarded by magnetic aspiration, and 500 μL of pH 8.5, 1M ethanolamine buffer is added to the magnetic beads, and incubated on a vertical mixer at 25°C for 10 minutes to block the unreacted activation sites on the surface of the magnetic beads. After the incubation, the blocking solution is discarded by magnetic aspiration. The magnetic beads are washed with 500 μL of DPBS-T buffer (containing 5 mM Mg 2+ The beads were washed four times with 0.02% Tween20 in DPBS buffer (pH 7.0), and then resuspended in PBS to obtain magnetic beads coupled with lung cancer serum, labeled as MB-LC, and stored at 4°C.

[0094] 2) Carboxyl magnetic beads coupled to HSA human serum albumin (counter-screening magnetic beads)

[0095] The method is the same as step 1), except that the homogenized lung cancer serum is replaced with HSA human serum albumin (purchased from Beijing Solebow Technology Co., Ltd.), the volume of the coupled carboxyl magnetic beads is changed to 600 μL, the amount of coupled HSA human serum albumin is 150 μg, and other operations are the same. The magnetic beads coupled with HSA human serum albumin are marked as MB-HSA.

[0096] 3) Library dissolution and denaturation treatment

[0097] Take the Library10 random single-stranded nucleotide library (1OD) synthesized by Bio-Industry, centrifuge at 12000rpm for 10min, centrifuge the library to the bottom of the tube, dissolve it to 5μM with DPBS buffer, mix it and dispense it into PCR tubes for denaturation treatment. The treatment process is as follows: set the PCR instrument program to 95℃ for 10min. The purpose of this step is to unwind the folded chain, then keep it at 4℃ for 5min, and then equilibrate it to room temperature. Then add Mg to the library. 2+ (final concentration of 5 mM), BSA (final concentration of 0.5 mg / mL), and HSDNA (final concentration of 0.05 mg / mL) were mixed to provide the ionic environment required by ssDNA, reduce nonspecific binding, and increase competitiveness. This is the library after denaturation treatment.

[0098] 4) Positive screening

[0099] Take 50μL MB-LC in an EP tube, remove the supernatant by magnetic suction, wash twice with 200μL DPBS-T buffer, remove the supernatant by magnetic suction, add 280μL of 5μM treated random ssDNA library and mix it, place it on a vertical mixer and incubate at room temperature for 120min. After the incubation, remove the supernatant by magnetic suction, retain the magnetic beads, wash the magnetic beads 4 times with 200μL DPBS-T buffer, and remove the supernatant by magnetic suction. Add 100μL ultrapure water, boil in a water bath for 10min, collect the supernatant, and mark it as "elution-LC". Prepare the secondary ssDNA library using the nucleic acid molecules in elution-LC as templates, and repeat the method to adjust the screening pressure for 7 rounds of screening.

[0100] 5) Reverse screening

[0101] Take 45 μL MB-HSA in an EP tube, remove the supernatant by magnetic suction, wash twice with 200 μL DPBS-T buffer to remove the supernatant, add 100 μL of the secondary ssDNA library obtained in the 7th round of screening, mix well and incubate on a vertical mixer at room temperature for 60 minutes. At the end of incubation, collect the supernatant as the ssDNA library used for the 8th round of positive screening. Use this method to adjust the screening pressure and screen 8 to 11 rounds. After each round of reverse screening, wash the remaining magnetic beads with 200 μL DPBS buffer 4 times, add 100 μL ultrapure water, boil in water for 10 minutes, collect the supernatant (i.e., reverse screening supernatant), and mark it as elution-HSA.

[0102] 6) Preparation of secondary library

[0103] 10 μL of elution-LC in step 4) and elution-HSA in step 5) were quantified by qPCR (real-time fluorescence quantitative PCR), and the screening process was monitored by calculating the number of molecules retained in each round. The results are shown in Figure 1 .

[0104] The nucleic acid molecules in step 4) elution-LC were used as templates and amplified by ordinary PCR. The method is as follows: 90 μL of template elution-LC was added to 900 μL of PCR mix, and 10 μL of ultrapure water was added for PCR amplification. The amplification conditions are as follows: 95°C pre-denaturation for 2 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, a total of 25 cycles, and stored at 4°C. The PCR raw materials in the PCRmix were prepared by dNTPs (P031-02) purchased from Novozymes and rTaq enzyme (R500Z) purchased from Takara Biotech.

[0105] The amplified dsDNA products were purified using commercial Tiandirenhe SA magnetic beads (SM017010) to prepare the secondary ssDNA library for the next round of screening. Prepare 80μLSA magnetic beads, wash them twice with 500μL DPBS-T buffer, discard the supernatant by magnetic suction, add 1000μL of PCR product, and then add 1 / 5 volume of 4M sodium chloride aqueous solution of its PCR product, incubate on a shaker at room temperature for 30min, and discard the supernatant by magnetic suction. Wash the magnetic beads three times with DPBS-T buffer, remove the supernatant and add 100μL of 40mM sodium hydroxide aqueous solution, incubate at room temperature for 3min, and then remove the magnetic beads by magnetic suction. Then add 4μL of 1M hydrochloric acid to the supernatant to neutralize the single strand, and then add 104μL of 2×DPBS buffer to dilute and neutralize the salt concentration, and finally obtain 208μL of the secondary library dissolved in 1×DPBS buffer, which can be used as the library for the next round of screening.

[0106] The magnetic bead method was repeated for 11 rounds of screening, each operation using the secondary library obtained in the previous operation as the starting nucleic acid library, and the operation was performed according to steps 3), 4), 5) and 6).

[0107] 7) Library affinity detection

[0108] The library enrichment was determined based on the qPCR Cq value of elution during each round of positive and negative screening. If the amount of template eluted from each round of positive screening increased (Cq advanced) with the increase in screening rounds and both the amplification curve and the melting curve reached a stable state, the screening was stopped. Figure 1 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. After the screening, the recognition ability of the DNA single-stranded library with MB-LC and MB-HSA was detected by flow cytometry. The specific operation was as follows:

[0109] The single-stranded libraries obtained in the 1st, 5th, 9th and 11th rounds of screening and the initial Library10 random single-stranded nucleotide library were selected for denaturation treatment: 40 μL of 400 nM single-stranded library was taken and Mg was added. 2+ (final concentration of 5mM), BSA (final concentration of 0.5mg / mL) were mixed, and 2μL MB-LC was added respectively, and incubated in the dark at 25℃ on a vertical mixer for 1h. Place on a magnetic stand, discard the supernatant, retain the magnetic beads, wash 4 times with 500μL DPBS-T buffer, and resuspend with it, and mark them as LC+Pool1, LC+Pool5, LC+Pool9, LC+Pool11, and LC+Random.

[0110] Under the same conditions, step 1) MB-LC was replaced by step 2) MB-HSA as a control, and the products were labeled LC-Pool1, LC-Pool5, LC-Pool9, LC-Pool11, and LC-Random.

[0111] Prepare 2 μL of carboxyl magnetic beads, discard the supernatant by magnetic aspiration, add 600 μL of DPBS-T buffer and resuspend as a blank control, marked as NC.

[0112] Then the above 11 samples were tested by flow cytometry. The flow cytometry graph is shown in Figure 2 shown.

[0113] Despite the challenges posed by the complexity of screening targets, Figure 2The flow fluorescence shift of the single-stranded library and the positive screening target in each round also showed an increasing trend with each round and had no binding with the initial random library, indicating that the nucleic acid molecules in the library that can bind to the positive screening target are effectively enriched. With the increase of screening rounds, the library's recognition ability for the target gradually increased, that is, the affinity gradually increased. At the same time, the flow fluorescence shift of the library and the control reverse screening target did not change, indicating that the library had no affinity for the reverse screening target, thus confirming the effectiveness of the reverse screening step and ensuring that the screened nucleic acid aptamers had good specificity.

[0114] 3. Analysis and identification of the nucleic acid aptamers obtained after screening: After high-throughput sequencing analysis of the obtained enriched library products, 10 candidate sequences with high abundance rankings and different families were selected and synthesized by Jinweizhi Biotechnology (Jiangsu) Technology Co., Ltd., which are nucleic acid aptamers having nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and they are named DH01, DH02, DH03, DH04, DH05, DH07, DH08, DH15, DH18, and DH20, respectively.

[0115] Example 2: Real-time fluorescence quantitative PCR (qPCR) detection of the binding ability of 10 candidate sequences to positive and negative screening targets

[0116] Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize nucleic acid aptamers DH01 (SEQ ID NO.1), DH02 (SEQ ID NO.2), DH03 (SEQ ID NO.3), DH04 (SEQ ID NO.4), DH05 (SEQ ID NO.5), DH07 (SEQ ID NO.6), DH08 (SEQ ID NO.7), DH15 (SEQ ID NO.8), DH18 (SEQ ID NO.9), and DH20 (SEQ ID NO.10).

[0117] The 10 candidate sequences were tested for their ability to bind to targets by forward and reverse screening using qPCR. The 10 synthetic nucleic acid aptamers were diluted to 100 nM 200 μL with DPBS buffer, rapidly denatured, and then Mg was added. 2+(final concentration is 5mM), BSA (final concentration is 0.5mg / mL), and mix well. Divide into two equal parts, and incubate with 10μL of MB-LC and MB-HSA prepared in step 1) and step 2) at room temperature for 1h, respectively. After incubation, wash 4 times with 200μL DPBS-T, 2min / time. Then add 100μL of ultrapure water to each, boil in a boiling water bath for 10min, collect the washing eluate, perform qPCR quantification, and repeat the experiment three times. The concentration of each eluate is calibrated according to the Cq value of each sample to evaluate the binding of the nucleic acid aptamer to the target, and select the nucleic acid aptamer with the strongest binding force to the positive screening target and the significant difference in binding with the reverse screening target. The smaller the Cq value, the more nucleic acid molecules are bound to the target. See the results. Figure 3 , showing that the number of nucleic acid molecules bound to the positive screening target magnetic beads of the 10 nucleic acid aptamers was greater than that bound to the reverse screening target magnetic beads, proving that the binding ability of the above 10 nucleic acid aptamers to lung cancer serum was higher than that to healthy serum, indicating that they may specifically bind to proteins that are differentially upregulated in lung cancer patients. Among them, the nucleic acid aptamer DH03 has the strongest binding ability to the positive screening target. Figure 4 It shows that the concentration of nucleic acid aptamer DH03 binding to the forward and reverse screening targets differs by more than 10 times, making it the nucleic acid aptamer with the best affinity.

[0118] Example 3: Identification of DH03's specific recognition target protein in lung cancer serum by streptavidin-biotin method

[0119] Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the biotin-modified nucleic acid aptamer DH03 (SEQ ID NO.3) and the biotin-modified Library10 library synthesized by Sangong. Non-cancer serum samples were provided by the Laboratory Department of Zhejiang Cancer Hospital.

[0120] Agarose gel bead blocking solution (5% BSA): Weigh 0.5 g BSA, dissolve in 10 mL DPBS-T buffer, shake and mix, and store at 4°C.

[0121] Protein blocking solution: Weigh 0.01 g BSA, dissolve in 10 mL DPBS-T buffer, shake and mix, and store at 4°C.

[0122] 10% SDS: Weigh 1 g of SDS dry powder, add 10 mL of ultrapure water to dissolve it, and store at 4°C.

[0123] 10% APS: Weigh 1 g of APS dry powder, add 10 mL of ultrapure water to dissolve it, and store at -20°C.

[0124] 10×Tris-Gly electrophoresis buffer: weigh 188g glycine, 30.2g Tris, and 10g SDS in a 1L beaker, add 900mL pure water, and dilute to 1L after complete dissolution. Dilute to 1× before use.

[0125] (1) Serum protein sample preparation. The biotin-labeled aptamer DH03 was fixed on the streptavidin-coated agarose gel beads by using the highly specific binding ability of streptavidin and biotin, and then incubated with serum protein. The protein bound by the aptamer DH03 was eluted by high temperature denaturation. The specific steps are as follows:

[0126] ① Preparation of streptavidin-coated agarose gel beads: Take 3 EP tubes, mark them as Random-LC (random library-lung cancer serum sample), DH03-LC (DH03-lung cancer serum sample), DH03-Health (DH03 non-cancer serum sample), add 100 μL of streptavidin-coated agarose gel beads to each tube, wash 3 times with DPBS-T buffer, centrifuge conditions: 4°C, 5 min, 5500 rpm; add 200 μL of agarose gel bead blocking solution to each EP tube, seal with sealing film, shake and mix, incubate at 4°C, horizontal shaker 80 rpm, incubate for 1 hour; after blocking, wash 5 times with DPBS-T buffer, 5500 rpm, 4°C, 5 min, centrifuge and place on ice;

[0127] ②Serum protein blocking: Prepare 800 μL of lung cancer serum sample and 400 μL of non-cancer serum sample, add two volumes of protein blocking solution to each sample, shake and mix, incubate at 4°C, 80 rpm on a horizontal shaker, and incubate for 1 hour;

[0128] ③ The blocked lung cancer serum samples were divided equally and incubated with the biotin-modified Library10 library and the nucleic acid aptamer DH03 at 4°C for 1 hour. The blocked non-cancer serum samples were operated in the same manner as above with the biotin-modified nucleic acid aptamer DH03. (The nucleic acid aptamers incubated with the above three samples were prepared at 1μM 100μL, and were incubated with serum after rapid denaturation treatment).

[0129] ④ The blocked protein-aptamer complex was incubated with the corresponding labeled blocked agarose gel beads at 4°C for 45 min. After the incubation, it was washed 5 times with DPBS-T at 2500 rpm, 3 min, and 4°C.

[0130] ⑤ Add 25 μL of 5× protein loading buffer (Biosharp Biotechnology Co., Ltd.) to each of the three samples, shake to mix, centrifuge, 100°C, 10 min, ice bath, 10 min, centrifuge, 2500 rpm, 3 min, 4°C, take 25 μL for sample loading.

[0131] (2) SDS-PAGE electrophoresis. Prepare 10% separation gel and 5% concentration gel for electrophoresis according to the formula shown in Table 3. Prepare the electrophoresis plate, use a pipette to slowly add the mixed separation gel along the inner surface of the long glass plate of the gel chamber to prevent the generation of bubbles, and pour it to 2-3 cm from the upper edge of the short glass plate, about 5 mL. Use a pipette to inject a small amount of water into the gel and leave it at room temperature to allow the separation gel to solidify. After the separation gel polymerizes, pour out the water on the surface of the gel and dry it with filter paper. Use a pipette to slowly add the mixed concentration gel along the inner surface of the long glass plate of the gel chamber to the top of the separation gel, insert a 1.5 mm sample comb, and wait until there is a clear line between the concentration gel and the separation gel, indicating that the gel has solidified, and prepare for electrophoresis. Correctly load the prepared SDS-PAGE into the electrophoresis tank, pour in 1×Tris-Gly electrophoresis solution, and gently pull out the comb to avoid damaging the gel holes. Use a 1 mL syringe to absorb the electrophoresis solution and gently clean the sample wells to clean up the broken gel remaining in the gel holes. Add the prepared samples into the wells in the order of 1: Random-LC (non-specific protein); 2: DH03-LC (target protein); 3: DH03-Health (protein extracted from non-cancer serum), and connect the power supply. First, perform electrophoresis at 60V. After the bromophenol blue band migrates to the lower separation gel, increase the voltage to 120V and continue electrophoresis. When the bromophenol blue front band runs to 1cm in the separation gel, stop electrophoresis, stain with Coomassie Brilliant Blue, wash with decolorizing solution until decolorization is complete, and then cut the separation gel.

[0132] (3) Mass spectrometry identification and proteomic analysis. The gel strips were sent to Guangke Ande Biotechnology Co., Ltd., and the protein mixture in the gel strip samples was analyzed and identified by combining in-gel enzymatic hydrolysis and high-resolution biological mass spectrometry technology. Combining the three sets of mass spectrometry data, the protein binding protein function and abundance of the protein-binding protein only present in the DH03-LC group were selected to select the transforming growth factor β-induced protein TGFBI (SEQ ID NO.12) abnormally expressed in tumors as a potential binding target of the nucleic acid aptamer DH03.

[0133] Table 3. SDS-PAGE recipe

[0134]

[0135] Example 4: Surface Plasmon Resonance (SPR) Detection of Affinity between Nucleic Acid Aptamer DH03 and TGFBI Protein

[0136] The nucleic acid aptamer DH03 (SEQ ID NO.3) was washed with DPBS buffer (containing 5 mM Mg 2+ ) were diluted to 500nM, 250nM, 125nM, 62.5nM, 31.25nM, and 15.625nM.

[0137] 1. Take 5 μg of TGFBI protein (C-His, Uniport ID: Q15582, purchased from ABclonal) and add it to 190 μL of 10 mM NaAC solution at pH 4.0 and mix well for use. Set up the coupling program: load the CM5 chip of the surface plasmon resonance instrument (GE Healthcare, model: Biacore 8K) into the chip slot, mix EDC and NHS in equal volumes, inject 105 μL to activate the chip, and the flow rate is 10 μL / min; couple the TGFBI protein dilution to the second channel on the surface of the CM5 chip as the experimental channel, with an injection volume of 190 μL and a flow rate of 10 μL / min; after the injection is completed, seal the chip with ethanolamine, inject 129 μL, and the flow rate is 10 μL / min. The amount of TGFBI protein coupled is about 6400Ru.

[0138] Same operation as in channel 2: replace TGFBI protein with 10×His peptide (10 consecutive histidines, synthesized by GenScript Biotech Co., Ltd.), take 40μg His peptide protein and add it to 190μL 10mM NaAC solution at pH 4.5. The coupling procedure is the same, coupling His protein in channel 1 as the control channel.

[0139] 2. Detection: The aptamer DH03 samples diluted with different concentration gradients were passed through channels 1 and 2 in sequence. The procedure for each channel was as follows: injection 30 μL / min, time 3 min; DPBS buffer (containing 5 mM Mg 2+ ) was injected at 30 μL / min for dissociation, and the time was 3 min; 1.5 M NaCl aqueous solution was injected at 30 μL / min for regeneration, and the time was 45 s. Figure 5 shown.

[0140] Example 5: Surface Plasmon Resonance (SPR) Detection of Specificity of Nucleic Acid Aptamer DH03 and TGFBI Protein

[0141] TIM3 protein (Uniport ID: Q8TDQ0, purchased from Beijing Sino Biological Technology Co., Ltd.), CXCL5 protein (Uniport ID: P42830, purchased from Hangzhou Jingjie Biotechnology Co., Ltd.), CD5 protein (Uniport ID: P06127, purchased from ABclonal). Three random control sequences: RS-1, RS-2, and RS-3 were synthesized by Suzhou Genewise Biotechnology Co., Ltd., and the sequence information is shown in Table 4.

[0142] Table 4. Randomized control sequence information

[0143]

[0144] In this example, TIM3 protein, CXCL5 protein, and CD5 protein were used to replace TGFBI protein. The method of fixing TGFBI protein to the SPR chip for testing was the same as that in Example 4. TIM3 protein, CXCL5 protein, and CD5 protein were coupled to the second channel of three channels on the surface of the CM5 chip, and the coupling amounts were about 1600RU, 2000RU, and 5000RU, respectively. 2+ ) were diluted to 500 nM and injected sequentially.

[0145] The three random control sequences RS-1, RS-2, RS-3 and DH03 were respectively prepared by DPBS buffer (containing 5 mM Mg 2+ ) was diluted to 500 nM, and based on Example 4, affinity detection of TGFBI protein was performed by passing through channels 1 and 2 in sequence.

[0146] The affinity of nucleic acid aptamer DH03 with the above three proteins and the affinity of random control sequences RS-1, RS-2, RS-3 with TGFBI protein are shown in Figure 6 It can be seen that the nucleic acid aptamer DH03 does not bind to the three proteins, namely, TIM3 protein, CXCL5 protein, and CD5 protein, but only binds to the TGFBI protein. The TGFBI protein also does not bind to other random control sequences. It can be seen that the binding of DH03 to the TGFBI protein is highly specific.

[0147] Example 6: Detection of TGFBI protein at different concentrations by dot hybridization assay based on nucleic acid aptamer DH03

[0148] The dot hybridization experiment was performed on the biotin-modified nucleic acid aptamer DH03, and the steps were as follows:

[0149] (1) Dissolve TGFBI protein in DPBS buffer to 1.000 mg / mL, 0.500 mg / mL, 0.250 mg / mL, 0.125 mg / mL, 0.063 mg / mL, and 0.032 mg / mL as test samples; dissolve the control protein His peptide in DPBS buffer to 1.000 mg / mL as the control sample. Take a 3 cm × 1 cm test strip (nitrocellulose membrane, 0.2 μm, GE Amersham, catalog number 10600001), spot 2 μL of the test sample and control sample on the nitrocellulose membrane, and air dry for 40 minutes.

[0150] (2) Then, the test strip was placed in a 2 mL centrifuge tube, and 2 mL of DPBS buffer containing 10% BSA was added to soak it. The test strip was blocked on a shaker at room temperature for 1 hour. After blocking, it was washed with DPBS-T for 5 minutes. The washing was repeated 3 times and then aspirated clean.

[0151] (3) Transfer the test strip to a new 2 mL centrifuge tube and add 2 mL of DPBS buffer (containing 5 mM Mg 2+ ) was soaked with 500 nM biotin-modified aptamer DH03, incubated on a shaker at room temperature for 30 min, and washed with DPBS-T for 2 min after the incubation, and washed repeatedly for 3 times and then aspirated clean.

[0152] (4) Transfer the test strip to a new 2 mL centrifuge tube, add 2 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 incubation, wash it with DPBS-T for 2 min, repeat the washing 3 times, and aspirate it clean.

[0153] (5) Transfer the test strip to a clean PE glove, take an ECL color development kit (purchased from Beyotime Biotech), mix 50 μL of solution A and 50 μL of solution B, soak the surface of the test strip, and incubate in the dark for 5 min.

[0154] (6) Imaging system observation and photography: ImageQuant from GE Healthcare Life Sciences TM LAS 4000 digital imaging system takes pictures, the results are shown in Figure 7 .

[0155] Depend on Figure 7 It can be demonstrated that the nucleic acid aptamer DH03 can be used to detect 0.125 mg / ml TGFBI protein and does not bind to the control protein.

[0156] 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 that binds to lung cancer serum, characterized in that: The nucleic acid aptamer has a nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO.

10.

2. According to claim 1, a nucleic acid aptamer binding to lung cancer serum is found to bind to TGFBI protein in lung cancer serum, characterized in that: The nucleic acid aptamer has a nucleotide sequence that is at least 60% homologous to SEQ ID NO. 3 and binds to the TGFBI protein.

3. A nucleic acid aptamer binding to TGFBI protein according to claim 2, characterized in that: The nucleic acid aptamer has an RNA sequence transcribed from the nucleotide sequence shown in SEQ ID NO.

3.

4. A nucleic acid aptamer binding to TGFBI protein according to claim 2, characterized in that: The nucleic acid aptamer has a sequence obtained by truncating the nucleotide sequence shown in SEQ ID NO.

3.

5. A nucleic acid aptamer binding to TGFBI protein according to claim 2, characterized in that: The nucleic acid aptamer includes a sequence obtained by modifying a nucleotide sequence that has at least 60% homology to SEQ ID NO. 3 and binds to TGF BI protein.

6. A nucleic acid aptamer binding to TGFBI protein according to claim 5, characterized in that: The modification includes at least one of the following modification methods: (1) Phosphorylation; (2) Methylation; (3) Amination; (4) thiolation; (5) Isotopization; (6) Substituting sulfur for oxygen; (7) Replace oxygen with selenium.

7. The nucleic acid aptamer binding to TGFBI protein according to claim 2, characterized in that: The nucleic acid aptamer has a sequence obtained by modifying a nucleotide sequence that has at least 60% homology to SEQ ID NO.3 and binds to the TGFBI protein; 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 nucleic acid aptamer; (5) connecting digoxigenin to the aptamer; (6) connecting a nanoluminescent material to the nucleic acid aptamer; (7) Connecting a small peptide to the nucleic acid aptamer; (8) Connecting siRNA to the nucleic acid aptamer.

8. A derivative of a nucleic acid aptamer that binds to a TGFBI protein, characterized in that: The derivatives include at least one of the following: (1) a phosphorothioate backbone sequence derived from the nucleic acid aptamer backbone of claim 2; (2) A peptide nucleic acid sequence designed and synthesized from the nucleic acid aptamer according to claim 2.

9. Use of the nucleic acid aptamer according to any one of claims 2 to 7 and the nucleic acid aptamer derivative according to claim 8, characterized in that: Include at least one of the following: (1) Detection of TGFBI protein; (2) localization imaging of cells, tissues or living organisms expressing TGFBI; (3) capturing cells or exosomes expressing TGFBI; (4) Drug conjugation; (5) Targeted delivery of drugs to tumor cells; (6) Regulate TGFBI protein function; (7) preparing TGFBI protein detection reagent; (8) preparing imaging agents; (9) preparing drug carriers; (10) Preparation of anti-tumor drugs.

10. A screening method and target identification process based on lung cancer serum nucleic acid aptamers, characterized in that: The steps include: S1, synthesizing single-stranded ssDNA library and primers; S2, magnetic bead screening: lung cancer sera were screened for 7 rounds, and counter-screening was added in rounds 8-11. The conventional conditions for screening were carried out in ionic buffer; S3, high-throughput sequencing, selecting multiple aptamers with high abundance rankings and from different families; S4. Using real-time fluorescence quantitative PCR (qPCR) to verify the binding ability of multiple nucleic acid aptamers, and obtain the nucleic acid aptamer that best binds to lung cancer serum; S5. The optimal binding nucleic acid aptamer is used to identify the specific binding target in lung cancer serum through the streptavidin-biotin method.

Images