ATP (adenosine triphosphate) nucleic acid aptamer based on m6A epigenetic base modification and application of ATP nucleic acid aptamer

Abstract

The invention belongs to the field of biosensing detection, and particularly relates to an ATP (adenosine triphosphate) nucleic acid aptamer based on m6A epigenetic base modification and application of the ATP nucleic acid aptamer. The invention provides a nucleic acid aptamer and a nucleic acid aptamer sensor. On the basis of recognition and binding specificity of a nucleic acid aptamer probe to a target, a single-point methylation closed switch type functional probe is constructed, and living cell and living body imaging detection of endogenous demethylase is realized. Taking ATP and an aptamer thereof as an example, m6A methylation modifies key sites of the aptamer so as to influence the binding stability between the aptamer and a ligand, after endogenous FTO demethylation, the aptamer recovers active sites, can be rapidly bound with ATP and is driven to allosteric to release fluorescence, and in-situ instant detection of FTO is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor detection, and specifically relates to a method based on m 6 A. Epigenetic base-modified ATP nucleic acid aptamer and its application. Background Art

[0002] N6-methyladenine (m 6 A) refers to the methylation modification of the sixth nitrogen atom of adenine A, which is one of the most common epigenetic mechanisms in eukaryotic cells. 6 A modification plays a key role in regulating mRNA maturation, translation, and degradation. 6 A methylation modification plays different roles in the occurrence and development of tumors. 6 A methylation modification changes dynamically and is reversible. The main regulatory factors include methyltransferases (writers), demethylases (erasers) and m 6 A methylation reader protein (readers) three parts. 6 FTO (fat and obesity-associated protein), one of the A demethylases, its abnormal expression is the basis of the pathogenesis of various human diseases (such as acute myeloid leukemia AML, melanoma, breast cancer and cervical cancer, etc.), which also indicates that FTO can be used as a new diagnostic biomarker and become a therapeutic target for diseases including tumors.

[0003] However, due to the small size and chemical inertness of the N6-methyl group, the m 6 The detection of A demethylase activity in cells remains challenging. Therefore, only a few methods have been reported for analysis. These include detection of demethylated DNA / RNA products using HPLC or MALDI-TOF mass spectrometry, gel electrophoresis of enzyme-digested substrates, fluorescence analysis of fluorescently labeled single-stranded DNA substrates, m 6 Although these methods can analyze FTO, many of them are limited by poor sensitivity, low throughput, and offline ensemble analysis out of the living cell environment, ignoring cell-to-cell heterogeneity and losing key dynamic information of living cells. At the same time, to date, the real-time analysis of m in living cells has not been achieved. 6There are few methods for detecting the activity of FTO demethylase, and there are problems such as poor stability and low reaction efficiency, which lead to the inability to achieve long-term stable analysis or dynamic information lag. In addition, affected by the regulation of the circulatory system and microenvironment in the body, the necessity of in vivo detection cannot be ignored. However, the current analytical methods have not been applied at the in vivo level, which seriously hinders the research of these clinically important enzymes. Therefore, it is urgent to develop a method that is fast, sensitive, simple and can trace the activity of FTO enzyme in real time in vivo, and apply it to the screening of FTO inhibitors. Summary of the invention

[0004] The invention idea of ​​the present invention is: the present invention develops a m 6 A site-specific modified ATP aptamer sensor is used to realize a new method for in vivo FTO detection. The aptamer sensor (ASP sensor) consists of an aptamer, a poly-T linker and a closed chain to form a complete hairpin structure. The Cy3 fluorescent group and its corresponding quenching group BHQ2 are modified at the 5' and 3' ends of the ASP sensor, respectively, so that the initial fluorescence signal is in a quenched state. First, the recognition and binding ability of the ASP sensor for ATP is controlled by the m at the key base site of the aptamer. 6 A methylation modification inhibits the binding energy of aptamer to ATP, making it less than the free energy of binding to the closed chain. 6 When the A-ASP sensor is delivered into cells by liposomes, the endogenous demethylase FTO in the cytoplasm 6 m in A-ASP sensor 6 A undergoes oxidative demethylation to restore the recognition and binding activity of the ASP sensor to endogenous ATP. The binding energy of the aptamer with ATP is greater than the free energy of binding with the closed chain, causing the probe to undergo conformational changes and restore fluorescence, thereby achieving imaging detection of the endogenous demethylase FTO.

[0005] The purpose of the first aspect of the present invention is to provide a nucleic acid aptamer.

[0006] The second aspect of the present invention aims to provide a 6 A modified nucleic acid aptamer sensor.

[0007] The third aspect of the present invention aims to provide the nucleic acid aptamer of the first aspect of the present invention, the m 6 Application of A-modified nucleic acid aptamer sensor in product preparation.

[0008] The fourth aspect of the present invention aims to provide a reagent.

[0009] The fifth aspect of the present invention aims to provide a kit.

[0010] The sixth aspect of the present invention aims to provide an imaging system.

[0011] The seventh aspect of the present invention aims to provide a method.

[0012] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0013] The first aspect of the present invention provides a nucleic acid aptamer, characterized in that:

[0014] The sequence of the nucleic acid aptamer is shown in SEQ ID NO.1.

[0015] The 10th and / or 23rd adenine of the nucleic acid aptamer has m 6 A modification.

[0016] Preferably, the 10th adenine of the nucleic acid aptamer has m 6 A modification.

[0017] A second aspect of the present invention provides a 6 A modified nucleic acid aptamer sensor, the m 6 A modified nucleic acid aptamer sensor comprises a nucleic acid aptamer sequence, a poly-T sequence, and a closed chain sequence; the m 6 The two ends of the A-modified nucleic acid aptamer sensor have a fluorescent group and a quenching group.

[0018] Preferably, the m 6 The A-modified nucleic acid aptamer sensor has a nucleic acid aptamer sequence, a poly-T sequence, and a closed chain sequence from the 5' end to the 3' end.

[0019] Preferably, the length of the poly-T sequence is 3 to 25 bases, more preferably, 4 to 20 bases.

[0020] In some embodiments of the present invention, the length of the poly-T sequence is 12 T base sequences.

[0021] Preferably, the length of the closed chain sequence is 4 to 10 bases.

[0022] Preferably, the closed strand sequence is reversely complementary to the 5' end of the nucleic acid aptamer sequence.

[0023] In some embodiments of the present invention, the closed chain sequence has 6 bases, which complementarily pair with the first 6 bases at the 5' end of the nucleic acid aptamer sequence.

[0024] Preferably, the m6 The 5' and 3' ends of the A-modified aptamer sensor have stable bases.

[0025] Preferably, the m 6 The stable base at the 5' end of the A-modified nucleic acid aptamer sensor is complementary to the stable base at the 3' end.

[0026] Preferably, the number of the stable bases is 1 to 3.

[0027] Preferably, the stabilizing base is selected from G or C.

[0028] In some embodiments of the present invention, the number of the stable base is one, wherein the stable base at the 5' end is C, and the stable base at the 3' end is G. Similarly, the stable base at the 5' end may be G, and the stable base at the 3' end may be C.

[0029] Preferably, the fluorescent group is selected from at least one of FAM, HEX, ROX, TAMRA, JOE, Cy3, Cy5, and VIC.

[0030] Preferably, the quenching group is selected from at least one of Dabcyl, BHQ1, BHQ2, BHQ3, MGB and TAMRA.

[0031] Preferably, the nucleic acid aptamer sequence has m 6 A modification;

[0032] Preferably, the sequence of the nucleic acid aptamer is as shown in SEQ ID NO.1, and the 10th and / or 23rd adenine of the nucleic acid aptamer has m 6 A modification.

[0033] In the second aspect of the present invention, the design idea of ​​the nucleic acid aptamer sensor is to introduce a poly-T sequence and a closed chain sequence so that the sensor itself forms a looped hairpin structure. When there is no ATP, the closed chain sequence is complementary to the 5' end sequence of the nucleic acid aptamer sequence, so that the fluorescent groups at both ends of the sensor are close to the quenching group, so that the initial fluorescence is in a quenched state. When there is ATP, the binding energy of the nucleic acid aptamer sequence with ATP is greater than the free energy of binding with the closed chain, so that the sensor configuration changes, the fluorescent group is separated from the quenching group, and the sensor restores the fluorescence signal to achieve the detection of ATP.

[0034] Based on this idea, further, the m 6 The design idea of ​​the A-modified aptamer sensor is to modify the aptamer sequence based on the aptamer sensor. 6A modification blocks the binding site between the initial aptamer sequence and ATP, and the aptamer sequence cannot bind to ATP. 6 The introduction of A methylation modification changes the function of the aptamer sensor from detecting ATP to detecting demethylase. When there is no demethylase, the aptamer sequence cannot bind ATP and cannot change its conformation, and the initial fluorescence is in a quenched state. When there is a demethylase, the demethylase can remove the m on the aptamer sequence. 6 A is modified so that the sensor recovers its original ability to bind ATP, and based on the design idea of ​​the nucleic acid aptamer sensor in the second aspect of the present invention, the sensor recovers the fluorescence signal in the presence of ATP, thereby realizing the detection of demethylase.

[0035] Those skilled in the art can select a suitable fluorescent group and match it with a corresponding quenching group according to the specific detection band of the detection instrument. The fluorescent group and the quenching group are respectively located at the two ends of the sensor, and their specific positions and directions will not be a limitation of the present invention.

[0036] Furthermore, those skilled in the art can, based on the above ideas, make corresponding modifications to other aptamer sequences that can bind to ATP. Due to experimental cost and cycle factors, the applicant has only modified the sensor of one aptamer sequence (SEQ ID NO.1), but it can be expected that other ATP aptamer sequences can also be modified based on this idea to obtain other aptamer sensors with different sequences. The present invention provides a preferred aptamer sequence that binds to ATP, and other sequences that bind to ATP can also be expected to achieve similar or identical effects of the present invention. The specific sequence of the aptamer should not be a limitation of the present invention. For example, since the ATP nucleic acid aptamer contains the important G4 structure that binds to the ligand, this method performs m6A methylation on the A base near G4, thereby interfering with the binding of the aptamer to the ligand, reducing the spatial matching and interaction between the two, and ultimately reducing the binding ability of the two. It can be inferred that this method can be applied to aptamers containing G4 structures, such as the thrombin aptamer TBA and the nucleolin aptamer AS1411, both of which contain G4 structures that bind to the ligand. The m6A modification near these G4 structures can be predicted to interfere with their binding ability, so that the aptamer sequence in ASP can be replaced with TBA or AS1411.

[0037] The third aspect of the present invention provides the nucleic acid aptamer of the first aspect of the present invention, the m 6 A modified nucleic acid aptamer sensor is used in the preparation of a product, wherein the product is used for any one of a1) to a5):

[0038] a1) Detection of demethylase;

[0039] a2) screening demethylase activators;

[0040] a3) screening for demethylase inhibitors;

[0041] a4) Cell imaging;

[0042] a5) Imaging of subcellular structures.

[0043] Preferably, the demethylase comprises at least one of fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5).

[0044] Preferably, the demethylase activator includes a substance that increases the expression of demethylase, a substance that increases the activity of demethylase, and a substance that delays the decomposition of demethylase.

[0045] Those skilled in the art can complete the screening of the above activators or inhibitors by designing a corresponding experimental system. For example, a small amount of demethylase is added to an in vitro experimental system, and the candidate molecules are screened through the system. If the addition of a candidate molecule increases the fluorescence signal, it means that the candidate molecule has the function of activating the activity of the demethylase; or a corresponding cell system is designed, a specific screening system is designed, and candidate molecules that increase the activity of the demethylase, increase the expression of the demethylase, and delay the decomposition of the demethylase are screened.

[0046] Preferably, the demethylase inhibitor refers to a substance that reduces the expression level of demethylase, a substance that reduces the activity of demethylase, or a substance that promotes the decomposition of demethylase.

[0047] Similarly, those skilled in the art can screen the corresponding inhibitors based on the screening ideas of activators.

[0048] In the third aspect of the present invention, the product includes but is not limited to at least one of a reagent, a kit, a system, and a chip.

[0049] The cell imaging refers to tracking the dynamic changes of ATP or demethylase in the living cell environment based on real-time fluorescence response changes. Subcellular structure imaging refers to tracking the dynamic changes of ATP or demethylase in the subcellular structure of the living cell environment based on real-time fluorescence response changes.

[0050] The subcellular structure includes at least one of a cell nucleus, a mitochondria, an endoplasmic reticulum, a Golgi apparatus, a lysosome, a vesicle system, and a cell membrane.

[0051] The fourth aspect of the present invention provides a reagent, comprising the nucleic acid aptamer of the first aspect of the present invention, and / or the m 6 A modified nucleic acid aptamer sensor.

[0052] The fifth aspect of the present invention provides a kit, comprising the nucleic acid aptamer of the first aspect of the present invention, and / or the m 6 A modified nucleic acid aptamer sensor.

[0053] Preferably, the kit further comprises a buffer.

[0054] Preferably, the buffer comprises at least one of PBS, TBS, Tris-HCl and HBSS.

[0055] Preferably, the kit comprises a positive standard template and a negative standard template. Preferably, the positive standard template is a quantitative ATP or a quantitative demethylase standard sample.

[0056] Preferably, the negative standard template is a standard sample that does not contain ATP or demethylase.

[0057] Preferably, the positive standard template is used to determine whether the detection system can successfully complete the detection.

[0058] Preferably, the negative standard template is used to determine whether there is no sample contamination in the detection system.

[0059] Preferably, the kit further comprises instructions.

[0060] Preferably, the instructions record the specific information of each reagent, usage instructions and other operating instructions.

[0061] A sixth aspect of the present invention provides an imaging system, comprising the following components:

[0062] 1) Sample detection component;

[0063] 2) Data processing components;

[0064] 3) Result output component.

[0065] Preferably, the sample detection component comprises the reagent of the fourth aspect of the present invention, and / or the kit of the fifth aspect of the present invention.

[0066] Preferably, the sample detection component comprises a detection instrument.

[0067] Preferably, the detection instrument includes at least one of a laser microscope and a living body imager.

[0068] The seventh aspect of the present invention provides a method according to any one of c1 to c2), comprising the step of using the reagent according to the fourth aspect of the present invention and / or the kit according to the fifth aspect of the present invention.

[0069] c1) Methods for detecting ATP for non-diagnostic and therapeutic purposes;

[0070] c2) Methods for detecting demethylases for non-diagnostic and therapeutic purposes.

[0071] The beneficial effects of the present invention are:

[0072] The present invention provides a nucleic acid aptamer, namely a nucleic acid aptamer sensor. Based on the recognition and binding specificity of the nucleic acid aptamer probe to the target, a single-site methylation closed switch type functional probe is constructed to achieve the living cell and living body imaging detection of endogenous demethylase. Taking ATP and its aptamer as an example, m 6 A methylation modifies the key sites of the aptamer to affect the binding stability between the aptamer and the ligand. After demethylation of endogenous FTO, the aptamer restores the active site, can quickly bind to ATP, and is driven to release fluorescence allosterically, realizing in situ instant detection of FTO. This method has the following advantages:

[0073] 1. This report is the first to achieve in situ imaging detection of FTO at the living level, and it is also the first to use deoxyribonucleotide (DNA) aptamer probe to detect FTO enzyme activity;

[0074] 2. The improved nucleic acid aptamer probe is a single-stranded DNA hairpin probe. Compared with the reported RNA probe, it is more convenient to synthesize, has a stable structure, is not easy to degrade, and has good biosafety;

[0075] 3. Through computer-assisted molecular simulation, accurate molecular docking can be achieved and the effects of methylation modification on thermodynamics and kinetics can be studied;

[0076] 4. A single methyl modification can cause a huge difference in the binding energy of the ATP aptamer;

[0077] 5. High sensitivity, good accuracy, and detection limit as low as 0.3nM;

[0078] 6. Good specificity, no specific response to other endogenous proteins and small molecule compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0080] Figure 1 This is a top view of the three-dimensional structure of the Aptamer1 model.

[0081] Figure 2The three-dimensional structure and distance of the two quadruplexes of the Aptamer1 model.

[0082] Figure 3 is m 6 Effect of A modification on ATP binding at A10 and A23 sites; a) Binding interactions between A10 and b) A23 sites in Aptamer1 and ATP molecules; c) Modeling of ATP binding interactions at A10 site in Aptamer2 and d) A23 site in Aptamer3; e) Changes in the binding energy of A10 and f) A23 with ATP before and after mA modification during kinetic simulation.

[0083] Figure 4 Conformational changes of the Aptamer-ATP complex at different time points during the kinetic simulation.

[0084] Figure 5 Schematic diagram of ASP sensor detecting FTO protein.

[0085] Figure 6 A / m 6 Comparison of thermodynamic differences of A-ASP sensor; (A) ASP sensor response to different ATP concentrations, (B) A / m 6 The relative fluorescence intensity differences between A-ASP sensors.

[0086] Figure 7 A / m 6 Comparison of A-ASP sensor dynamics differences.

[0087] Figure 8 A / m 6 A-ASP sensor response to FTO; (A)m 6 A-ASP sensor analyzes the final fluorescence change values ​​of FTO protein at different concentrations; Inset: m 6 Linear curve of A-ASP sensor detecting FTO protein; (B) A / m 6 The response of A-ASP sensor to FTO protein.

[0088] Fig. 9 is m 6 The responsiveness of A-ASP sensor to different proteins and small molecules in cells.

[0089] Fig.10 is m 6 The detection results of A-ASP sensor for FTO inhibitors; (A) m activated by FTO 6A-ASPsensor evaluates the inhibition curve of FTO inhibitors; (B) Chemical structure of small molecule and IC calculated from the inhibition curve in (A) 50 value.

[0090] Fig.11 For gel electrophoresis experiments 6 Stability results of A-ASP sensor in cellular environment.

[0091] Fig.12 For fluorescence experiment analysis m 6 The responsiveness of A-ASP sensor to different types of cell lysates.

[0092] Fig.13 is m 6 The imaging results of FTO in cells by A-ASP sensor. The scale bar is 20μm.

[0093] Fig.14 for Fig.13 The cell fluorescence images in the image were analyzed by ImageJ software to obtain the distribution of Cy3 fluorescence signals, ***P<0.001.

[0094] Fig.15 is m 6 Biosafety assessment of A-ASP sensor. PBS was selected as the control group. The scale bar is 200 μm.

[0095] Fig.16 is m 6 In vivo imaging of A-ASP sensor in MCF-7 tumor-bearing nude mice; a) Whole-body fluorescence imaging of MCF-7 tumor-bearing mice after injection of different systems: (i) m 6 A-ASP sensor, (ii) m after FTO inhibitor pretreatment 6 A-APSsensor, (iii)m 6 A-CtrlDNA; b) Ex vivo imaging of tumors and major organs (He: heart, Li: liver, Sp: spleen, Lu: lung, Ki: kidney) of mice 12 hours after injection; c) Quantification of fluorescence in tumor areas in b); error bars are from n=2 experiments. DETAILED DESCRIPTION

[0096] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0097] The reagents involved in the present invention are derived from: all DNA chains and modifications are synthesized by Shanghai Bioengineering Technology Co., Ltd., and the DNA sequence is shown in Table 3; recombinant human FTO is purchased from Active Motif Company of the United States; rhein and isonicotinic acid are purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); Entacapone is purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China).

[0098] Related instruments include: NIKON AX laser confocal microscope; IVIS Lumina III in vivo imaging system (PerkinElmer, MA, USA).

[0099] Example 1 Based on computer-aided molecular design (CAMD) 6 Effect of A methylation modification on ATP-aptamer

[0100] 1. Sequence design of ATP-aptamer

[0101] In this example, aptamer1 was used as the research object, and m was modified at different positions. 6 The three aptamer sequences of A, the base sequence of single-chain ATP-aptamer and the m at different sites 6 A methylation modified sequences are shown in Table 1.

[0102]

[0103] 2. Model construction

[0104] Based on Discovery Studio2.5 Client, Aptamer1 was initially modeled. Two G4 structures were first determined, and then complementary end sequences and intermediate sequences were constructed, and then combined to form the three-dimensional structure of Probe1. Finally, the constructed model was optimized in a water environment using Gromacs2018 dynamics software. The optimization process is mainly divided into three steps: a. The obtained structure is vacuum optimized; b. Adding a physiological environment, the Aptamer1 quadruplex structure of the module key is subjected to a 200ps equilibrium dynamics simulation to obtain the optimized Aptamer1 structure.

[0105] The final structures of Aptamer1 are Figure 1 After optimization, the quadruplex in the three-dimensional structure of Aptamer1 was quantitatively analyzed, see Figure 2 ;Depend on Figure 2It can be seen that in the structure of Aptamer1, the distance between G5, G8, G18, G21 and G6, G9, G19, and G22 is very short due to the interactive double hydrogen bonds, and they alternately form a quadruple structure, which makes Aptamer1 exist stably.

[0106] 3. Molecular docking simulation study

[0107] 3.1 Ligand preparation

[0108] First, download the sdf file of the small molecule of ATP from the PDB database; second, use the MM2 method to optimize the ligand and save it in mol2 format for the next docking study. In ADT, assign AD atom type to ATP, set the root and rotatable bond number of the ligand molecule, add hydrogen and charge, and finally merge non-polar hydrogen and save it in pdbqt format.

[0109] 3.2 Receptor preparation

[0110] Discovery studio2.5 Client was used to construct a model of the Aptamer1 structure containing two G4s, and then A10 and A23 were methylated to obtain Aptamer2 and Aptamer3. Aptamer4 was methylated on A10 and A23 at the same time to form three new aptamer three-dimensional structures, and dynamic optimization was performed to obtain reasonable conformations for the next step of research. Finally, Aptamer1, Aptamer2, Aptamer3, and Aptamer4 were hydrogenated, charged, and assigned atomic types in Autodock Tools and saved as pdbqt structure files.

[0111] 3.3 Molecular docking

[0112] Aptamer1, Aptamer2, Aptamer3, and Aptamer4 were used as receptors and ATP molecules were used as ligands. The grid energy calculation file was established using the AutoDockTools software. The molecular docking calculations of DNA aptamers Aptamer1, Aptamer2, Aptamer3, and Aptamer4 with ATP were performed using the Autodock program. The results are shown in Table 2. The lower the binding energy, the stronger the binding interaction between the two. The 100 conformations output by the docking results were clustered to obtain the optimal binding conformation. As shown in Table 2, compared with the A10 binding site of Aptamer2 and the A23 binding site of Aptamer3, the binding energy of Aptamer1 with ATP at A10 and A23 is relatively low, that is, the binding stability is higher; this may be due to the methylation of the A10 of Aptamer2 and the A23 of Aptamer3, which affects their binding with ATP, resulting in decreased binding stability; in the process of docking calculation of the binding with ATP, Aptamer4 directly performs molecular docking calculation with ATP through the complex of Aptamer2 and ATP to obtain its binding energy at A23, which is used to evaluate the interaction of the two molecules with Aptamer4 at the same time. The results show that the binding energy generated by docking is basically similar to that in the previous Aptamer2 and Aptamer3. Therefore, there is no mutual influence between the binding of ATP in A10 and A23 in Aptamer4, and its binding energy is also lower than that of Aptamer1, and the binding stability is also worse.

[0113] Table 2 Docking scoring results of Aptamer and ATP using AutoDock

[0114]

[0115]

[0116] In order to gain a deeper understanding of the binding of the aptamer to ATP during the kinetic simulation, the binding energy of the aptamer to ATP in the four complex systems of Aptamer1-A10-ATP, Aptamer2-ATP, Aptamer1-A23-ATP and Aptamer3-ATP was quantitatively analyzed. The results are shown in Figure 2. Figure 3As shown. The lower the binding energy, the greater the binding stability. After the kinetic simulation equilibrium, the binding energy between the Aptamer2-A10 aptamer and ATP is around -4400 kJ / mol, with low binding energy and good binding stability, and it is relatively stable throughout the kinetic simulation stage; compared with Aptamer1-A10, the binding energy between the Aptamer2 aptamer and ATP is only around -100 kJ / mol during the entire kinetic simulation, with higher binding energy and lower binding stability. At the same time, there are obvious fluctuations in the binding energy, which may be caused by its local dissociation. Therefore, the methylation of A10 of the aptamer Aptamer1 may lead to a decrease in the binding stability of ATP, thereby reducing its activity.

[0117] After the dynamics simulation is balanced (30-50ns) ( Figure 4 ), the binding energy of Aptamer1-A23 and Aptamer3 aptamers with ATP is basically similar, both around -200kJ / mol. Among them, the binding energy of Aptamer3 with ATP after kinetic simulation equilibrium is about -150kJ / mol, while the binding energy of Aptamer3 with ATP is about -260kJ / mol, with lower binding energy and better binding stability. Therefore, although the methylation of A23 in Aptamer1 will not cause the dissociation of ATP and the aptamer, it will still reduce its binding stability and lead to a decrease in its activity.

[0118] Example 2m 6 Feasibility study of A-ASP sensor for detecting FTO in vitro

[0119] 1. Aptamer Switch Probe sensor (ASP sensor) construction and ATP driver verification

[0120] ATP aptamer was used as the experimental object. To increase the stability of aptamer, a pair of CG bases were added to its 5' and 3' ends. Then, poly-T linker and closed chain were connected to its 3' end to form a closed ASP sensor. The 5' end was modified with fluorescent group Cy3, and the 3' end was modified with quenching group BHQ1. Figure 5 Based on the high specificity of the aptamer recognition core, we modified the A base of its active center with N6-aminomethyl (m 6 A) to block its activity, and then under the action of FTO protease, m 6 A is demethylated and aptamer activity is restored, thereby constructing an FTO-activated ASP sensor, which can realize the detection of FTO protein and the screening of inhibitors in vitro and in vivo.

[0121] The sequence of ASP sensor is shown in Table 3.

[0122] Table 3 ASP sensor sequence and m 6 A methylation modification site

[0123]

[0124]

[0125] 2. m 6 A-ASP sensor response to ATP

[0126] Thermodynamic fluorescence verification: A-ASP (indicates no m 6 A modified ASP) and m 6 A-ASP (final concentration 200 nM) and different concentrations of ATP (final concentrations of 5, 10, 20, 50, 80, 100, 200, 500, 800, 1000, 2000 μM) were mixed and fixed to 50 μL with Tris-HCl buffer (10 mM Tris-HCl, 6 mM MgCl2), and the fluorescence intensity was measured (excitation wavelength 535 nm, emission spectrum range 545 nm to 700 nm). Figure 6 As shown in A, at a certain ATP concentration, m 6 A methylation modification has a great influence on the binding energy of ASP sensor. 6 The fluorescence difference between A-ASP sensors is the largest, with a minimum of 7 times. Figure 6 As shown in B.

[0127] Kinetic fluorescence verification: A-ASP and m 6 A-ASP (final concentration 200 nM) was added to Tris-HCl buffer (10 mM Tris-HCl, 6 mM MgCl2) and the volume was adjusted to 48 μL. The real-time fluorescence intensity was measured, and then ATP (2 μL, final concentration 500 μM) was added to observe the real-time fluorescence changes within 0-30 s. Figure 7 As shown in the figure, after adding ATP at the 10s node, A-ASP and m 6 A-ASP can respond quickly, showing good kinetic response effects, and still showing huge differences in fluorescence intensity.

[0128] 3. m 6 Verification of the response effect of A-ASP sensor on FTO

[0129] Detection limit fluorescence verification: First, m 6A-ASP-sensor (sensor shown in SEQ ID NO.6, 2μM), 283μM (NH4)2Fe(SO4)2·6H2O, 300μM α-KG, 2mM L-ascorbic acid, 50μg / mL BSA and different concentrations of FTO were mixed and fixed to 20μL with 50mM HEPES (pH 7.0) to make the final concentration of FTO in the system (0, 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, 1000nM). Then incubate at 37°C for 3h. Then add 5mM EDTA and incubate at 95°C for 5min to terminate the reaction.

[0130] Take the above FTO after demethylation 6 A-ASP-sensor (sensor shown in SEQ ID NO.6, 5 μL, final concentration 200 nM) was reacted with ATP (500 μM) in 50 μL Tris-HCl buffer (10 mM Tris-HCl, 6 mM MgCl2), and the fluorescence intensity was measured immediately. Figure 8 As shown in Figure A, the fluorescence intensity of the system gradually increases with the increase of FTO protein content (from 0 to 1000 nM), and the linear detection range obtained from the corresponding calibration curve is 2 to 20 nM. Based on the traditional 3σ / s criterion (σ represents the standard deviation of 10 blank samples, and s is the slope of the calibration curve), the detection limit of FTO reaches 0.3 nM, which is comparable to or even better than the sensitivity of existing FTO detection methods. Figure 8 As shown in B, the m 6 The fluorescence intensity of A-ASP sensor recovered to the equivalent level of A-ASP sensor, and after demethylation, the fluorescence intensity increased to 9.75 times the original level, which was a significant difference.

[0131] Specific fluorescence verification: First, m 6 A-ASP-sensor (sensor shown in SEQ ID NO.6, 2 μM), 283 μM (NH4)2Fe(SO4)2·6H2O, 300 μM α-KG, 2 mM L-ascorbic acid, 50 μg / mL BSA and different endogenous proteins (20 U mL –1 APE1,200nM thrombin,50U mL –1UDG, 50μg / mL BSA) or small molecule compounds (5mM GSH, 100μM H2O2, 2mM glucose) were mixed and fixed to 20μL with 50mM HEPES (pH 7.0) and incubated at 37℃ for 3h. Then 5mM EDTA was added and incubated at 95℃ for 5min to terminate the reaction. 6 A-ASP (5 μL, final concentration 200 nM) was reacted with ATP (500 nM) in 50 μL Tris-HCl buffer (10 mM Tris-HCl, 6 mM MgCl 2 ), and the fluorescence intensity was measured immediately.

[0132] The results are as follows Fig. 9 As shown in the figure, by exploring the interference of some common proteins and small molecules in cells on the probe, it was found that m 6 A-ASP sensor is only responsive to FTO protein, indicating that 6 A-ASP sensor is not only sensitive to FTO protein, but also has good selectivity.

[0133] 4. m 6 Screening and detection of FTO inhibitors by A-ASP sensor

[0134] FTO (1 μM) was pretreated with small molecule inhibitors (entacapone, rhein, and negative inhibitor isonicotinic acid) at different concentrations (nM) in 50 μL HEPES buffer solution (50 mM, pH 7.0, 283 μM (NH4)2Fe(SO4)2·6H2O, 300 μM α-KG, 2 mM L-ascorbic acid, 50 μg / mL BSA) and incubated at 37 °C for 1 h. 6 A-ASP-sensor (sensor shown in SEQ ID NO.6, final concentration 2 μM), incubated at 37°C for 3 h. Repeat the above termination reaction steps and react with ATP in Tris-HCl buffer, and measure the fluorescence intensity. IC 50 The relative activities from different inhibitor concentrations (2, 5, 10, 20, 50, 100, 200 μM) were obtained by nonlinear regression fitting in GraphPad Prism 7.0. 50 value.

[0135]

[0136] F 抑制剂 : Add FTO protein and inhibitor; F 无抑制剂 : Add FTO protein without inhibitor; F 空白: No FTO protein and inhibitor were added.

[0137] like Fig.10 As shown, in the presence of small molecule inhibitors, m 6 The demethylation capacity of A demethylase, m 6 A-ASP sensor cannot be activated and cannot recognize and bind to ATP to achieve conformational change. The fluorescence signal is low, and the fluorescence intensity value is negatively correlated with the concentration of the inhibitor. The inhibitory effect of the inhibitor is explained by the fluorescence intensity of the system. The inhibitory effect is expressed by the relative activity of the FTO protein, and the inhibitory curve of the inhibitor is obtained ( Fig.10 As expected, the results showed that the activity of FTO decreased significantly with the increase of the concentration of inhibitors (entacapone and rhein), while the non-inhibitor (isonicotinic acid) had little effect on the activity of FTO at the same concentration, indicating that m 6 A-ASP sensor can be effectively used for the screening and detection of FTO small molecule inhibitors.

[0138] Example 3m 6 Feasibility study of A-ASP sensor for FTO live cell imaging detection

[0139] 1. m 6 A-ASP sensor stability verification in cell environment

[0140] 1 μM m 6 A-ASP-sensor (sensor shown in SEQ ID NO.6) was incubated in an equal volume of MCF-7 cell lysate at 37°C for 0-10 h, and then stability analysis was performed by polyacrylamide gel electrophoresis. Fig.11 As shown, within 10 hours, m 6 The A-ASP sensor showed almost no degradation and maintained good stability, proving that it can withstand the complex intracellular environment for a certain period of time, laying the foundation for the system to enter cells and detect intracellular FTO proteins.

[0141] 2. Detection of FTO protein in cell lysate

[0142] Obtaining cell lysate: MDA-MB-231, MCF-7 and MCF-10A cells were seeded in 6-well plates. When the cells grew to 90% density, the cells were digested with trypsin and washed twice with ice PBS. The cells were lysed by repeated freeze-thaw method, centrifuged at 4°C, 12000rpm for 10min, and the supernatant was obtained to obtain the protein. The extracted protein was quantified using the BCA kit. Then 100nMm 6A-ASP was reacted with 0.8 mg / mL cytoplasmic lysate in HEPES reaction buffer for 3 h, added to Tris-HCl buffer system, and reacted with ATP at room temperature. The fluorescence intensity of the solution was measured using a fluorescence spectrometer.

[0143] like Fig.12 As shown, m 6 A-ASP sensor can respond to FTO protein in cell lysate. 6 A-ASP sensor (sensor shown in SEQ ID NO.6) reacted, and breast cancer cells (MDA-MB-231 and MCF-7) produced stronger fluorescence signals than normal breast cells (MCF-10A), indicating that the FTO protein and ATP content in breast cancer cells were higher than those in normal cells, which was consistent with the literature reports. This proved that the system can also achieve quantitative analysis of FTO protein in complex physiological environments.

[0144] 3. Based on m 6 FTO imaging analysis in living cells using A-ASP sensor

[0145] The mammalian FTO overexpression plasmid pCDNA3.1-FTO is used to up-regulate the FTO protein in cells. FTO-siRNA is used to down-regulate the FTO protein in cells.

[0146] The siRNA sequences used are as follows:

[0147] siRNA targeting FTO (si FTO): 5'-UCUCACAAGCAGCGGCUAUUU-3' (SEQ ID NO. 10);

[0148] Negative control siRNA (si Ctrl): 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO. 11);

[0149] (1) Downregulation of FTO protein using siRNA:

[0150] Plant the cells in a 12-well plate and grow them for 12 hours, with a density of about 60%. All items used for transfection of siRNA are enzyme-free experimental consumables. According to the ratio of 50μL Opti-MEM mixed with 2μL, 20μM siRNA, and 50μL Opti-MEM mixed with 3μL Lipo 3000, calculate the amount of Opti-MEM to be mixed according to the amount of siRNA to be transfected. Add the two mixed solutions together, mix gently, let stand at room temperature for about 10 minutes, and add 100μL siRNA-Lipo 3000 mixed solution to the 12-well plate to be transfected. After incubation in the incubator for 12 hours, change to complete culture medium and continue to culture for 48-72 hours before use for subsequent experiments.

[0151] (2) Upregulation of FTO protein using plasmid:

[0152] Plant the cells in a 12-well plate and grow them for 12 hours to a density of about 80%. Calculate the amount of Opti-MEM required according to the amount of plasmid to be transfected, by mixing 50 μL Opti-MEM with 1 μg of plasmid (pcDNA3.1-FTO) and 50 μL Opti-MEM with 3 μL Lipo 3000. Add the two mixed solutions together, mix gently, let stand at room temperature for about 10 minutes, and add 100 μL of the mixed solution to the 12-well plate to be transfected. After incubation in the incubator for 12 hours, replace with complete medium and continue to culture for 24-48 hours before use in subsequent experiments. Use commercial Lipofectamine 3000 (Invitrogen) to transfect 80nFTO siRNA or 2μg / mL FTO plasmid, and transfect according to the instructions.

[0153] The full-length FTO plasmid was purchased from Wuhan Miaoling Biotechnology Co., Ltd. and constructed into the pcDNA3.1(+) mammalian vector with an N-terminal FLAG tag. siRNA was synthesized by Shanghai Bioengineering, and the sequence was: siFTO: 5'-UCUCACAAGCAGCGGCUAUUU-3' (SEQ ID NO.10); Control siRNA (Ctrl-siRNA): 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO.11).

[0154] MCF-7 cells pretreated with pCDNA3.1-FTO (FTO up-regulation), pCDNA3.1 (up-regulation control group), si FTO (FTO down-regulation), si Ctrl (down-regulation control group), and untreated MDA-MB-231, MCF-7, and MCF-10A cells were subjected to the above m 6A-ASP transfection step, incubated at 37°C for 4 h. Then the cell nuclei were stained with Hoechst 33342 for 10 min, washed four times with PBS, and finally photographed with a laser confocal scanning microscope.

[0155] The results are as follows Fig.13 and 14 As shown, use m 6 A-ASP sensor analyzes the different expression levels of FTO protein in cells and monitors the level of FTO in cells. Compared with the two cancer cells (MDA-MB-231, MCF-7), normal breast cells (MCF-10A) only produce weaker fluorescence signals, indicating that the expression level of FTO protein in cancer cells is higher than that in normal cells, which is consistent with previous reports. These results show that the m 6 The A-ASP sensor imaging system can determine the expression difference of FTO protein based on the strength of the fluorescence signal to distinguish normal cells from tumor cells. In addition, by monitoring MCF-7 cells with different FTO expression levels, the m 6 The reliability and stability of A-ASP sensor in monitoring intracellular FTO content. By introducing siRNA that silences FTO and plasmids that express FTO, endogenous FTO protein was downregulated and upregulated, respectively. As expected, the fluorescence of MCF-7 cells pretreated with siRNA was significantly reduced, while the fluorescence of MCF-7 cells pretreated with plasmids was significantly increased. This indicates that FTO protein is indeed present in MCF-7 cells and activates m 6 A-ASP sensor, indicating that the m 6 The A-ASP sensor imaging system can monitor different FTO levels in cells, revealing the m 6 A closed m 6 The accuracy and sensitivity of A-ASP sensor for detecting FTO protein in cells.

[0156] Example 4m 6 Feasibility study of A-ASP sensor for FTO in vivo imaging detection

[0157] The experimental animals used in the present invention were BALB / c female nude mice (4–6 weeks old) from Zhuhai Baishitong Company, with permission from Shenzhen Top Biotechnology Co., Ltd. and reviewed by the Ethics Committee of Shenzhen Top Biotechnology Co., Ltd. (approval number: TOPGM-IACUC-2023-0074).

[0158] 1. Biosafety Verification

[0159] m 6PBS (200 μL) and untreated PBS (200 μL) co-incubated with A-ASP sensor (SEQ ID NO.6) and Lipo 3000 were injected into BALB / c-nu athymic nude mice via the tail vein. BALB / c-nu mice were euthanized 48 hours later, and the main organs including heart, liver, spleen, lung and kidney were obtained for H&E staining.

[0160] The results are as follows Fig.15 As shown, m 6 A-ASP sensor did not cause substantial changes to the various organs of mice and had good biosafety.

[0161] 2. In vivo FTO protein imaging analysis

[0162] Construction of nude mouse MCF-7 subcutaneous tumor model: MCF-7 breast cancer cells (1×10 7 cells / mouse); tumors grew to 70 mm 3 Afterwards, the mice were divided into three groups and injected peritumorally with m-Lipo3000 at a dose of 5 nmol / kg. 6 A 10 -ASP-Sensor (SEQ ID NO.6) and carried out in vivo imaging experiments. Nude mice bearing subcutaneous tumors (MCF-7 tumors) were used as animal models. 6 The A-ASP sensor mixture was injected peritumorally (refer to the reagent instructions). As time passed after injection, the fluorescence signal was monitored at different time points using an in vivo imaging system ( Fig.16 a). In the injection of m 6 Obvious fluorescence signals were observed in the tumor area of ​​A-ASPsensor (group i). The fluorescence signal reached the maximum value only 2 h after injection. 6 Before A-ASPsensor, mice were pre-injected with 50uL 10mM entacapone (group ii). Compared with group i, the fluorescence intensity decreased significantly after 2 hours, proving that m 6 A-ASPsensor has high reliability in vivo. In addition, injection of m 6 A 10 -CtrlDNA (SEQ ID NO.9) tumor region (group iii) to verify m 6 The specificity of A-ASPsensor for FTO in vivo. 6 A-CtrlDNA treated mice obtained weak fluorescence signals, indicating that m 6 A-ASPsensor is specific for FTO in vivo. 6In vivo imaging of A-ASPsensor and inhibitor pre-treatment groups confirmed that this was activated by FTO. Fig.16 As shown in middle b, further quantitative analysis of in vivo imaging experiments showed that m 6 Intact mice treated with A-ASPsensor showed a 3.5-fold higher fluorescence signal than mice pretreated with FTO inhibitor 12 h after injection. 6 Compared with mice treated with A-CtrlDNA, a 5.4-fold higher fluorescence signal was obtained. 6 The A-ASPsensor has high stability and specificity for high-contrast FTO imaging in vivo.

Claims

1. A nucleic acid aptamer, characterized in that: The sequence of the nucleic acid aptamer is shown in SEQ ID NO.1; The 10th and / or 23rd adenine of the nucleic acid aptamer has m 6 A modification.

2. A m 6 A modified nucleic acid aptamer sensor, characterized in that: The m 6 A modified nucleic acid aptamer sensor includes a nucleic acid aptamer sequence, a poly-T sequence, and a closed chain sequence; The m 6 The two ends of the A-modified aptamer sensor have a fluorescent group and a quenching group; Preferably, the m 6 The modified aptamer sensor A has aptamer sequence, poly-T sequence, and closed chain sequence from 5' to 3' end; Preferably, the length of the poly-T sequence is 3 to 25 bases; Preferably, the length of the closed chain sequence is 4 to 10 bases; Preferably, the closed strand sequence is reversely complementary to the 5' end of the nucleic acid aptamer sequence; Preferably, the m 6 The 5′ and 3′ ends of the A-modified aptamer sensor have stable bases; Preferably, the m 6 The stable base at the 5' end of the A-modified aptamer sensor is complementary to the stable base at the 3' end; Preferably, the number of the stable bases is 1 to 3; Preferably, the stabilizing base is selected from G or C; Preferably, the nucleic acid aptamer sequence has m 6 A modification.

3. The m according to claim 2 6 A modified nucleic acid aptamer sensor, characterized in that: The m 6 The sequence of the modified nucleic acid aptamer is shown in SEQ ID NO.

1. 6 The 10th and / or 23rd adenine of the modified nucleic acid aptamer has m 6 A modification.

4. The nucleic acid aptamer according to claim 1, or the m according to any one of claims 2 to 3 6 A modified nucleic acid aptamer sensor is used in the preparation of a product, wherein the product is used for any one of a1) to a5): a1) Detection of demethylase; a2) screening demethylase activators; a3) screening for demethylase inhibitors; a4) Cell imaging; a5) Imaging of subcellular structures.

5. The use according to claim 4, characterized in that: The demethylase comprises at least one of obesity-related protein and AlkB homolog 5.

6. The use according to claim 4 or 5, characterized in that: The product includes at least one of a reagent, a kit, a system, and a chip.

7. A reagent comprising the nucleic acid aptamer according to claim 1 and / or the m according to any one of claims 2 to 3. 6 A modified nucleic acid aptamer sensor.

8. A kit comprising the nucleic acid aptamer according to claim 1 and / or the m according to any one of claims 2 to 3. 6 A modified nucleic acid aptamer sensor; Preferably, the kit further comprises a buffer; Preferably, the buffer comprises at least one of PBS, TBS, HBSS and HEPES.

9. An imaging system comprising the following components: 1) Sample detection component; 2) Data processing components; 3) Result output component; Preferably, the sample detection component comprises the reagent according to claim 7 and / or the kit according to claim 8; Preferably, the sample detection member comprises a detection instrument; Preferably, the detection instrument includes at least one of a laser microscope and a living body imager.

10. The method according to any one of c1 to c2), comprising the step of using the reagent according to claim 7 and / or the kit according to claim 8; c1) Methods for detecting ATP for non-diagnostic and therapeutic purposes; c2) Methods for detecting demethylases for non-diagnostic and therapeutic purposes.

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