Sirtuin 2 aptamer and its application

Abstract

The present invention provides an aptamer for Sirtuin 2 and its application. The nucleotide sequence of the aptamer is shown in SEQ ID NO. 1, which solves the technical problems of poor stability and high cost of current antibodies that recognize Sirtuin 2.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and specifically discloses an aptamer of sirtuin 2 and applications thereof. Background Art

[0002] Silent information regulator 2 (SIRT2) is a key member of the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase family of proteins, known as sirtuins. It regulates a variety of biological processes, including cell proliferation and apoptosis, DNA damage, and inflammatory responses, by deacetylating substrates. Establishing a rapid and cost-effective method for detecting SIRT2 protein in cells is crucial for studying its specific role in these biological processes.

[0003] Currently, SIRT2 detection methods primarily rely on antigen-antibody interactions, including immunofluorescence, Western blot, and enzyme-linked immunosorbent assay (ELISA). However, antibodies are susceptible to temperature and pH changes, exhibit poor stability, are prone to inactivation and denaturation, and have high production costs. Currently, there is a lack of rapid and cost-effective detection methods for SIRT2.

[0004] Aptamers, also known as aptamers or aptamers for short, are specific nucleic acid sequences that have been obtained through repeated in vitro screening and can bind to target molecules with high affinity and specificity. They are essentially single-stranded deoxynucleic acid or ribonucleic acid (DNA / RNA), and the base length is generally 10 to 80 nt. Aptamers can be screened by the systematic evolution of ligands by exponential enrichment (SELEX) method. Aptamers have a specific three-dimensional structure, and the nucleotides mainly form specific structures including hairpin structures, stem-loop structures, three-chain hybridization and G tetramers through hydrogen bonds, van der Waals forces, electrostatic interactions, stacking between aromatic groups and bases, hydrophobic and lipophilic interactions, etc., and then bind to and interact with the corresponding targets, affecting the structure and function of the targets. Almost all reported aptamers have high affinity for the target, and their dissociation constant (K d Aptamers are also known as "chemical antibodies" due to their high affinity, specificity, low immunogenicity, low cost, and excellent stability. Aptamers therefore hold broad application prospects in a variety of fields, including food and environmental safety testing, medical bioassays, tumor diagnosis, and targeted therapy. Aptamer-based fluorescent probes or biosensor detection methods have attracted considerable attention due to their ability to effectively address the shortcomings of conventional protein detection methods.

[0005] However, the number of protein aptamers that have been screened and can be effectively used is very limited, and the detection objects of target proteins are mainly concentrated on a few targets related to disease diagnosis, such as thrombin, alpha-fetoprotein, and carcinoembryonic antigen. At present, there is no SIRT2 aptamer. Summary of the Invention

[0006] The first object of the present invention is to provide an aptamer for Sirtuin 2, which solves the technical problems of poor stability and high cost of current antibodies recognizing Sirtuin 2.

[0007] A second object of the present invention is to provide an application of a sirtuin 2 aptamer in a solution to achieve rapid detection of sirtuin 2 in a solution or cell lysate.

[0008] The third purpose of the present invention is to provide an aptamer of sirtuin 2 for use in the in situ detection of sirtuin 2 in living cells, so as to realize in situ imaging detection of sirtuin 2 in cells, thereby solving the technical problem that the currently commonly used immunofluorescence imaging method is difficult to perform live cell imaging.

[0009] Compared with the prior art, the present invention has at least the following advantages and positive effects:

[0010] The present invention provides an aptamer for sirtuin 2 and its application. The aptamer can be used to quickly detect the concentration of sirtuin 2 in a solution and can stably identify sirtuin 2 in cells, thereby performing in situ imaging of sirtuin 2 in living cells with excellent imaging effect, providing an economical and highly feasible method for in situ imaging detection of sirtuin 2 in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0012] Figure 1 Schematic diagram of affinity characterization of candidate aptamers in the embodiments of the present invention;

[0013] Figure 2 Schematic diagram of affinity determination and secondary structure of p-45 among the candidate aptamers in the examples of the present invention;

[0014] Figure 3This is a linear regression graph of the fluorescence detection value of the aptamer sensor versus the SIRT2 protein concentration in Experimental Example 1 of the present invention;

[0015] Figure 4 These are in situ imaging images of SIRT2 protein in three different cell lines using the aptamer sensor in Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0016] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0017] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to specific embodiments.

[0018] The present invention provides an aptamer for sirtuin 2, the nucleotide sequence of which is shown in SEQ ID NO. 1. Secondly, the present invention provides an aptamer for sirtuin 2 for use in detecting sirtuin 2 in solution. Finally, the present invention provides an aptamer for sirtuin 2 for use in in situ detection of sirtuin 2 in living cells.

[0019] Example

[0020] SIRT2 aptamer screening:

[0021] The present invention adopts the magnetic bead-SELEX method to screen and obtain SIRT2 aptamers.

[0022] Initial library random sequence:

[0023] 5'-ATTGGCACTCCACGCATAGG-N(40)-CCTATGCGTGCTACCGTGA A-3' (N is A / T / C / G random deoxynucleotide).

[0024] Screening Process:

[0025] First, dilute the initial library (1 OD) to a final concentration of 10 μM using Dulbecco's phosphate-buffered saline (DPBS). Heat the solution at 95°C for 10 minutes, place it in an ice-water bath for 5 minutes, and equilibrate it to room temperature. Next, take 50 μL of magnetic beads and add 200 μL of DPBS buffer. Mix the suspension thoroughly, then fish the beads on a magnet. Aspirate the supernatant and repeat this wash three times. Mix 50 μL of 0.1 M N-hydroxysuccinimide (NHS) and 50 μL of 0.4 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), add the solution to the beads, and incubate at room temperature for 20 minutes. Fish the beads on a magnet, aspirate the supernatant, and wash the beads once with 200 μL of DPBS buffer.

[0026] The target protein SIRT2 was diluted with sodium acetate solution (pH 4.5) to a final concentration of 0.15 mg / mL in a total volume of 100 μL. This protein solution was then added to the magnetic beads from the previous step and incubated at room temperature for 30 minutes. The beads were then magnetized, the supernatant discarded, and the beads were blocked with 100 μL of 1 M ethanolamine for 10 minutes. The beads were then magnetized, the supernatant discarded, and the beads were washed three times with 200 μL of DPBS buffer to obtain magnetic beads-SIRT2 (MB-SIRT2). Finally, the denatured aptamer library solution was added to the MB-SIRT2 beads and incubated at room temperature for 45 minutes. The beads were magnetized, the supernatant discarded, and the beads were washed with 200 μL of DPBS buffer three times. The wash was repeated three times. 200 μL of DPBS buffer was added, the suspension was mixed, and the protein was denatured by heating in a 100°C water bath for 10 minutes. The beads were then magnetized, and the supernatant was collected for elution.

[0027] In the first round of screening, SIRT2 was used as the target protein. In the second round of screening, bovine serum albumin (BSA) was introduced as a counter-screening protein. The supernatant solution that did not bind to the counter-screening protein after incubation was collected as the aptamer library for the second round of positive screening. A total of 15 rounds of screening were performed.

[0028] Library amplification:

[0029] Conventional PCR amplification was performed on the aptamer secondary library solution obtained in the first round of screening. 2 mL of PCRmix solution was thawed and centrifuged, then added to a 10 mL centrifuge tube. The solution was then added to the elution and vortexed to mix thoroughly. 100 μL was then dispensed into eight PCR tube strips. The PCR amplification program was as follows: 95°C for 1 minute, 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by 25 cycles of 95°C for 30 seconds, and finally a 5-minute hold at 72°C.

[0030] For the second and subsequent secondary libraries, perform emulsion amplification. Thaw 2 mL of PCR mix and centrifuge, then add it to a 50 mL centrifuge tube. Add the eluent and vortex the tube to thoroughly mix. Add 8 mL of ePCR microdroplet generator oil and vortex for 1-2 minutes. Aliquot 100 μL of the vortexed emulsion into eight PCR tube strips. The emulsion PCR amplification program is as follows: 95°C for 1 minute, 95°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute. Repeat 25 cycles starting with the second 95°C for 1 minute, and then hold at 72°C for 5 minutes.

[0031] Single-stranded DNA secondary library preparation:

[0032] The amplified library was concentrated to approximately 100 μL and added to an equal volume of 2× TBE urea loading buffer. Mix by inversion and centrifugation. Heat at 95°C for 10 minutes and cool on ice for 5 minutes. The treated library was separated by 8% denaturing polyacrylamide gel electrophoresis (300V). After electrophoresis, the gel was removed and the green fluorescent band of interest was cut out with a blade under a handheld UV lamp. The band of interest was cut into pieces and placed in a 2 mL centrifuge tube. DPBS buffer was added and the tube was in a boiling water bath for 10 minutes. Centrifuge at 12,000 rpm for 1 minute. The supernatant was concentrated to approximately 100 μL. The solution was dialyzed against DPBS buffer at 4°C overnight.

[0033] ELISA method to determine the affinity of the secondary library:

[0034] The aptamer secondary library obtained from rounds 3-15 of screening was diluted to 1 μM. The forward and reverse primers linked to Biotin were diluted to 10 μM. PCR amplification was performed using 10 μL of the secondary library, 40 μL of each forward and reverse primer, 1 mL of PCR Super Mix, and 910 μL of enzyme-free water. The PCR amplification program was as follows: 95°C for 2 min, 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s. This cycle was repeated 35 times, starting with the second 95°C for 1 min, and then held at 72°C for 5 min. The amplified product was recovered and concentrated to approximately 100 μL with n-butanol. An equal volume of 2× TBE-urea loading buffer was added, mixed, centrifuged, heated at 95°C for 10 min, and then ice-cooled for 5 min. The library was then separated by electrophoresis on an 8% denaturing polyacrylamide gel at 300 V. After electrophoresis, the gel was removed and stained in 3× GelRed solution for 30 min at room temperature. Remove the gel and, under a handheld UV lamp, recover the red fluorescent band at 80 nt. After fragmenting the target band, place it in a 2 mL centrifuge tube and add DPBS buffer. Boil in a boiling water bath for 10 minutes, centrifuge at 12,000 rpm for 1 minute, and concentrate the supernatant to approximately 100 μL. Dialyze this solution against DPBS buffer overnight at 4°C. Dilute SIRT2 to 10 μg / mL using protein coating buffer. Pipette 100 μL of the solution onto a high-affinity protein-coated microtiter plate. Apply a sealing film and refrigerate at 4°C overnight. The next day, wash the protein-coated wells three times with 100 μL of 1× wash buffer, soaking and washing for 1 minute each time. Add 100 μL of 1% BSA-PBS buffer and block at 37°C for 1 hour. Wash three times with 100 μL of 1× wash buffer, soaking for 1 minute each time. Then, add 100 μL of the 5'-biotin-linked sublibrary and incubate at 37°C for 90 minutes to allow specific binding of SIRT2 protein to the sublibrary. Wash three times with 100 μL of 1× wash buffer, soaking for 1 minute each time. Add 100 μL of ABC working solution, seal the plate, and incubate at 37°C for 1 hour. Wash five times with 100 μL of 1× wash buffer, soaking for 1 minute each time. Add 90 μL of TMB substrate and incubate at 37°C in the dark for 15 minutes. Finally, add 100 μL of TMB stop solution to each well and measure the OD value at 450 nm using a microplate reader.

[0035] Secondary library sequencing and candidate adapters:

[0036] High-throughput sequencing was performed on eight representative secondary libraries. The sequencing results were arranged by the total number of sequences. Some sequences were enriched through screening. These sequences with enrichment trends were then subjected to homology analysis, and 12 DNA sequences with low homology were selected as candidate aptamers. The sequences of these 12 candidate aptamers are shown in Table 1.

[0037] Table 1 Aptamer sequences

[0038]

[0039]

[0040] Affinity characterization of candidate aptamers:

[0041] The affinity of 12 candidate aptamers to SIRT2 was characterized by ELISA. Figure 1 As shown in Figure 2, all 12 candidate aptamers have good affinity with SIRT2. Figure 2 The results showed that p-45 had the best affinity, with a dissociation constant of 116.7±30.5nM.

[0042] Test Example 1

[0043] SIRT2 aptamer sensor is used to detect different concentrations of SIRT2 in solution:

[0044] SIRT2 aptamer sensor solution (100 nM aptamer-50 μg / mL MnO2) was prepared using DMEM cell culture medium as a buffer and incubated in the dark for 10 minutes. SIRT2 protein was then added to final concentrations of 0, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, respectively. The solution was incubated in the dark at 37°C for 60 minutes to allow fluorescence recovery. Fluorescence intensity was measured using a fluorescence spectrophotometer with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The experiment was repeated three times.

[0045] The SIRT2 concentration was linearly regressed using the mean fluorescence intensity. Figure 3 As shown in the figure, the fluorescence intensity has a good linear relationship with the SIRT2 concentration. 2 The value is 0.98, indicating that this aptamer sensor can detect the concentration of SIRT2.

[0046] Test Example 2

[0047] SIRT2 aptamer sensors were used for in situ imaging of SIRT2 in three cell lines:

[0048] RSC96 cells, resuspended in complete DMEM medium supplemented with 10% fetal bovine serum, were seeded at a density of 5,000 cells / well in a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. Similarly, H9c2 cells and HUVECs were seeded at a density of 5,000 cells / well in a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours, the medium was aspirated, and 100 μL of aptamer (100 nM aptamer-50 μg / mL MnO2) was added to each well and incubated with the cells in a 37°C, 5% CO2 incubator for 4 hours. The medium containing the aptamer was aspirated, and the cells were stained with Hoechst solution at a concentration of 2 μg / mL for 10 minutes. The cells were then washed three times with PBS. Finally, the cells were imaged using a laser scanning confocal fluorescence microscope using the FITC and DAPI channels.

[0049] SIRT2 protein is widely expressed in RSC96 cells, H9c2 cells and HUVECs cells. Therefore, we incubated the aptamer sensor with the above three cell lines to observe whether it can perform in situ imaging of SIRT2 in the three cell lines. The results are shown in Figure 2. Figure 4 As shown, from Figure 4 After incubation with the sensor, strong green fluorescence signals were observed in the cytoplasm of each cell line, indicating that the aptamer sensor can be endocytosed by the cells for SIRT2 imaging detection. Therefore, the constructed aptamer sensor can be used to study the biological functions of SIRT2 in the three cell lines mentioned above.

[0050] In summary, the present invention provides a sirtuin 2 aptamer and its application:

[0051] This aptamer can be used to quickly detect the concentration of sirtuin 2 in a solution and can stably identify sirtuin 2 in cells, thereby performing in situ imaging of sirtuin 2 in living cells with excellent imaging effects, providing an economical and feasible method for in situ imaging detection of sirtuin 2 in living cells.

[0052] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

Claims

1. A Sirtuin 2 aptamer, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.1.

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