Fluorocarbon chain-mediated organelle-targeting aptamer in-cell screening method and application thereof

Abstract

The application discloses a fluorocarbon chain-mediated organelle-targeting nucleic acid aptamer intracellular in-situ screening method and application thereof. The method comprises the following steps: loading and self-assembling ssDNA library and fluorocarbon chain-modified capture sequences into nanoparticles, incubating and endocytosing cells, separating and purifying organelles, eluting and amplifying target binding sequences, and preparing secondary libraries, and the above steps are repeated for multiple times, so that the dominant sequences in the initial random library are enriched and gradually evolved into mitochondrion-targeting nucleic acid aptamers. The application is an in-situ screening method in living cells, and can obtain aptamers with stronger performance and more superior application potential in a complex intracellular microenvironment, and can be applied to screening of aptamers with specific subcellular structure or organelle targeting in mammalian cells. The screened aptamers can be used for preparing subcellular imaging molecular probes and organelle-targeting delivery tools, and can also be used for researches on organelle function regulation and organelle interaction.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biotechnology and nanomedicine, and relates to a fluorocarbon chain-mediated in-situ intracellular screening method for organelle-targeting nucleic acid aptamers and its application, for the discovery of functional nucleic acid molecules (such as aptamers) targeting specific subcellular structures or organelles. Specifically, this invention relates to a method for using a fluorocarbon chain-mediated self-assembly nanodelivery system of nucleic acid libraries to achieve efficient intracellularization and controlled release, and for in-situ screening of nucleic acid aptamers targeting organelles (such as mitochondria) within living cells (such as HeLa cells). Background Technology

[0002] Nucleic acid aptamers are a class of single-stranded DNA or RNA molecules obtained through exponential enrichment ligand system evolution technology. They can bind to various targets with high affinity and specificity. Due to their advantages such as convenient synthesis, ease of modification, and low immunogenicity, nucleic acid aptamers have shown broad application prospects in molecular diagnostics, targeted therapy, and biosensing.

[0003] As biomedical research moves into the subcellular level, developing specific molecular tools targeting key organelles such as mitochondria is crucial for understanding life processes and diagnosing and treating related diseases. However, existing technological approaches face significant challenges in obtaining nucleic acid aptamers that can function effectively in the natural intracellular environment and target specific organelles.

[0004] Currently, most nucleic acid aptamer screening is conducted in vitro. This method cannot reproduce the actual conformation, modification state, and complex microenvironment of target molecules within living cells. Therefore, aptamers obtained through traditional in vitro screening often exhibit reduced affinity and specificity, or even functional failure, when applied intracellularly, indicating a fundamental mismatch between the screening environment and the application scenario.

[0005] To achieve in situ intracellular screening, highly diverse random nucleic acid libraries must be delivered efficiently and intact to the cytoplasm, ensuring their stable intracellular presence and escape from endocytic vesicles to achieve sufficient contact with the target. However, naked nucleic acid libraries are difficult to spontaneously penetrate the cell membrane and are easily captured and degraded by endosomes / lysosomes or rapidly broken down by intracellular nucleases after entering the cell. Conventional nucleic acid delivery systems have the following shortcomings when applied to the delivery of large-capacity, highly diverse nucleic acid libraries: liposomes and cationic polymers have limited encapsulation and delivery efficiency, severely affecting the initial diversity and signal-to-noise ratio of screening; the inherent cytotoxicity of cationic materials can affect cell viability and interfere with the screening process; viral vectors or certain chemical vectors may trigger immune responses, interfering with normal cellular physiological states and affecting the reliability of screening; in addition, many delivery systems rely on passive endocytosis and lack active escape mechanisms, causing the library to remain in endosomes / lysosomes and eventually be degraded, failing to achieve in situ interaction with target organelles.

[0006] Given the aforementioned challenges, developing a novel delivery system that simultaneously achieves efficient loading of randomized libraries, low cytotoxicity, efficient cell internalization, and endosome / lysosome escape is crucial for overcoming the bottlenecks in intracellular in-situ screening technology. Fluorocarbon chains (FCs) are a class of alkane chains in which hydrogen atoms are replaced by fluorine atoms. The high bond energy of the carbon-fluorine (CF) bond makes fluorinated alkyl chains highly resistant to various chemical reagents, oxidants, and reducing agents, and less prone to chemical reactions. In recent years, FC materials have attracted widespread attention due to their unique physicochemical properties. FCs possess both hydrophobic and lipophobic properties, low surface energy, and excellent self-assembly tendency. Studies show that FC modification can drive the self-assembly of delivery systems (such as oligonucleotides and peptides) into more uniform and stable nanoparticles, thereby promoting their efficient internalization by cells. Simultaneously, this modification can create a spatial shield by forming a dense nanostructure, effectively protecting nucleic acids from degradation by intracellular nucleases and enhancing their stability. Furthermore, the rigid amphiphilic structure of fluorocarbon chains can interfere with membrane lipid dynamics in the acidic environment of endosomes, disrupting the stability of endosome / lysosomal membranes and thus achieving efficient escape. Although fluorocarbon chains have shown potential in improving the delivery efficiency of single nucleic acid sequences, their application in constructing self-assembled nanostructures for delivering large-scale random nucleic acid libraries, and further combining them with in situ intracellular screening techniques, has not yet been reported, leaving this field unexplored.

[0007] Taking mitochondria as an example, there is currently a lack of ideal tools for efficiently and specifically delivering functional molecules to mitochondria. Widely used targeting groups (such as triphenylphosphine, TPP) suffer from problems such as strong membrane potential dependence, potential cytotoxicity, and non-specific binding to non-target cellular structures. Although studies have used aptamers of known mitochondrial-related proteins (such as cytochrome C) to construct nanocarriers, these aptamers are not obtained through in-situ screening in living mitochondria, limiting their targeting efficiency and function. Direct in vitro screening from purified organelles faces technical obstacles such as the easy destruction of organelle structural integrity and the inability to simulate the in-situ interaction environment, making it difficult to obtain high-performance aptamers.

[0008] In summary, existing technologies lack an effective method for efficiently, stably, and biocompatiblely delivering nucleic acid libraries into the cytoplasm of living cells and, within a realistic physiological microenvironment, directly screening for nucleic acid aptamers with high affinity and specificity for specific subcellular organelles. This technological bottleneck also restricts the development of molecular tools targeting subcellular structures and their application in precision medicine. To overcome this bottleneck, this invention proposes and develops a novel screening scheme: Intracellular SELEX (hereinafter referred to as In-cell SELEX). This technology aims to deliver complete nucleic acid libraries into the cytoplasm of living cells through an innovative delivery system, directly screening for nucleic acid aptamers with high functionality for specific subcellular organelles within a realistic physiological microenvironment. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a highly efficient and stable intracellular in situ nucleic acid aptamer screening platform technology. Specifically, it is a method for screening nucleic acid aptamers targeting specific subcellular structures or organelles within living cells using a fluorocarbon chain-mediated nucleic acid library self-assembly nanodelivery system. Based on this nanodelivery system and screening method, the present invention has screened a group of nucleic acid aptamers with high affinity and specificity for HeLa cell mitochondria, showing promising application prospects.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] In a first aspect, this invention provides an intracellular in-situ screening method for organelle-targeting nucleic acid aptamers mediated by a fluorocarbon chain, comprising the following steps: loading an ssDNA library with a fluorocarbon chain-modified capture sequence and self-assembling it into nanoparticles, incubating them with cells for intracellular delivery; washing cells to remove surface-attached nucleic acids and then isolating and purifying the target organelles; eluting the ssDNA attached to the target organelles and using it as a template for PCR amplification; preparing the PCR-amplified dsDNA product into ssDNA for use in the next round of screening; repeating the above steps multiple times to gradually enrich the dominant sequences and identify candidate nucleic acid aptamers through cloning and sequencing. In other words, through multiple rounds of screening, dominant sequences with high affinity and specificity to the target are continuously enriched, gradually evolving into preferred nucleic acid aptamers.

[0012] In some embodiments, the ssDNA library consists of a central 40 nt random sequence and a flanking 20 nt constant sequence, as shown in SEQ ID NO: 8; In some embodiments, the fluorocarbon chain-modified capture sequence is a fluorocarbon chain-modified capture sequence FC-CS that is completely complementary to the constant sequence of the ssDNA library. The base sequence of the capture sequence FC-CS is shown in SEQ ID NO: 1, and its 5' and / or 3' ends are modified with multiple fluorocarbon chains (preferably 6-12 fluorocarbon chains), with disulfide bonds embedded between the fluorocarbon chains and the base sequence. Preferably, the fluorocarbon chains are selected from C 10 F 21 CH2CH2-、C8F 17 CH2CH2-, and C6F 13 At least one of CH2CH2-.

[0013] In some specific embodiments, the concentration of the nanoparticles is 10 nM to 10 μM.

[0014] In some specific implementations, the incubation time with cells is 4-6 hours.

[0015] In some specific embodiments, the initial dose of the ssDNA library is 1280–3130 pmol / 10⁻¹. 7 Each cell.

[0016] In some specific embodiments, the preparation method of the fluorocarbon chain modified trapping sequence FC-CS includes the following steps: Step 1: Synthesis of FC phosphoramide monomer 1H,1H,2H,2H-perfluoro-1-octanol, 1H,1H,2H,2H-perfluoro-1-decanol, or 1H,1H,2H,2H-perfluoro-1-dodecanol were dissolved in dry dichloromethane with N,N-diisopropylethylamine and 2-cyanoethyl-N,N-diisopropylphosphonamide chloroform, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed, an aqueous sodium bicarbonate solution was added to terminate the reaction. The organic phases were extracted with dichloromethane and combined. After drying with anhydrous sodium sulfate, the mixture was concentrated under reduced pressure. The residue was co-evaporated with acetonitrile and used directly for subsequent DNA synthesis. Step 2: Solid-phase synthesis of FC-CS The DNA was synthesized using a solid-phase phosphorus amide chemical method on an automated DNA synthesizer. Disulfide groups were introduced sequentially at the ends of the DNA as shown in SEQ ID NO: 8, and two three-armed phosphorus amide branch units were coupled to increase the number of linkage sites to 9. The DNA was then coupled with FC phosphorus amide monomer to form DNA modified with 9 fluorocarbon chains. Step 3: After synthesis, the oligonucleotides are cleaved from the solid-phase support, deprotected, purified, and identified.

[0017] In some embodiments, the cell is a mammalian cell.

[0018] Preferably, the mammalian cells are tumor cells, immune cells, nerve cells, epithelial cells (e.g., HEK293 cells) or mesenchymal cells.

[0019] In some embodiments, the tumor cells are HeLa cells, A549 cells, MCF-7 cells, HCT116 cells, or Jurkat cells.

[0020] In some embodiments, the target organelle is a specific subcellular structure or organelle of the cell.

[0021] In some embodiments, the specific subcellular structure or organelle is an organelle related to energy metabolism, an organelle related to protein synthesis and processing, an organelle related to material degradation, a structure related to genetic material storage and processing, or a region related to cell signal transduction.

[0022] Preferably, the specific subcellular structure or organelle is a mitochondrial, nucleus, endoplasmic reticulum, Golgi apparatus, or lysosome.

[0023] In some embodiments, the primer pair used for PCR amplification is an upstream primer with the sequence shown in SEQ ID NO: 2 and a downstream primer with the sequence shown in SEQ ID NO: 3, wherein the upstream primer is modified with a fluorescent group and the downstream primer is modified with biotin.

[0024] In a second aspect, the present invention provides a reagent or kit for screening nucleic acid aptamers according to the method described in the first aspect of the present invention, the reagent or kit comprising an ssDNA library and a fluorocarbon chain-modified capture sequence; wherein: The ssDNA library consists of a 40 nt random sequence in the middle and a 20 nt constant sequence on the flanks, as shown in SEQ ID NO: 8; The fluorocarbon chain-modified capture sequence is a fluorocarbon chain-modified capture sequence FC-CS that is completely complementary to the constant sequence of the ssDNA library. The base sequence of the capture sequence FC-CS is shown in SEQ ID NO: 1. Multiple fluorocarbon chains are modified at its 5' and / or 3' ends, and disulfide bonds are embedded between the fluorocarbon chains and the base sequence.

[0025] In some embodiments, the reagent or kit further includes a PCR amplification primer pair, wherein the primer pair is an upstream primer with the sequence shown in SEQ ID NO: 2 and a downstream primer with the sequence shown in SEQ ID NO: 3, wherein the upstream primer is modified with a fluorescent group and the downstream primer is modified with biotin.

[0026] Thirdly, the present invention provides a mitochondrial-targeting nucleic acid aptamer, the sequence of which is shown in any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

[0027] In some embodiments, the 5' or 3' of the aptamer contains at least one non-naturally occurring chemical modification.

[0028] In some embodiments, the chemical modification is selected from one or more modifications selected from phosphate groups, amino groups, thiol groups, biotin, digoxigenin, fluorescent labels, quenching groups, lipid molecules, polyethylene glycol molecules, and nucleotide analogs.

[0029] Fourthly, this application provides the application of the mitochondrial-targeting nucleic acid aptamer as described in the third aspect, the application being selected from the group consisting of: i) the application in the preparation of subcellular imaging probes; ii) the application in the preparation of mitochondrial-targeting delivery tools; iii) the application in the study of mitochondrial functional regulation; and iv) the application in the study of the interaction between mitochondria and other subcellular structures or organelles.

[0030] Compared with the prior art, the present invention has the following beneficial effects: 1) Pioneering a new screening paradigm: This invention is the first to combine a fluorocarbon chain-mediated self-assembled nanodelivery system with SELEX screening, successfully constructing an in-cell SELEX platform, realizing in-situ screening of aptamers in the real physiological environment of living cells, fundamentally solving the problem of environmental mismatch in traditional in vitro screening.

[0031] 2) Breakthrough in core delivery technology bottlenecks: By utilizing the unique dihydrophobicity, self-assembly capability, and membrane interference properties of fluorocarbon chains, the constructed nanoparticles can simultaneously achieve efficient loading, low-toxicity intracellular delivery, and controllable cytoplasmic release of large-scale random nucleic acid libraries, overcoming long-standing technical challenges such as difficulty in library internalization, easy degradation, and low escape efficiency.

[0032] 3) Superior aptamer tools have been obtained: The aptamers screened through this platform (such as the mitochondrial-targeting aptamer) have evolved in the complex intracellular microenvironment. Therefore, they exhibit higher binding stability, stronger functional activity and lower off-target effects when applied in cells. Their application potential is significantly better than that of aptamers obtained through in vitro screening.

[0033] 4) High scalability and platform value: The In-cell SELEX platform established in this invention is not dependent on specific cell types. By changing the cell and target organelle types, the method can be universally applied to screen various functional nucleic acid molecules targeting different subcellular structures, providing a powerful and universal molecular tool development platform for cell biology research and precision medicine. Attached Figure Description

[0034] Figure 1 This is a schematic diagram (B) of the fluorocarbon chain modified capture sequence (A) and its mediated nucleic acid library self-assembly in an embodiment of the present invention.

[0035] Figure 2 In this embodiment of the invention, the gel electrophoresis characterization (A), transmission electron microscopy characterization (B), and cytotoxicity test of the fluorocarbon chain-mediated self-assembled nucleic acid library nanoparticles are described.

[0036] Figure 3 In this embodiment of the invention, the cellular uptake (A), endosome / lysosome escape kinetics (B), endocytosis coefficient to PCC ratio (C), and dose-effect analysis (D) of the fluorocarbon chain-nucleic acid library nanoparticles are described.

[0037] Figure 4 This is a flowchart of screening nucleic acid aptamers based on In-cell SELEX in an embodiment of the present invention.

[0038] Figure 5 In this embodiment of the invention, the preferred method is to analyze the co-localization of aptamers and mitochondria within HeLa cells.

[0039] Figure 6 In this embodiment of the invention, the preferred method is the co-localization analysis of aptamers and lysosomes within HeLa cells.

[0040] Figure 7 In this embodiment of the invention, the preferred method is the co-localization analysis of the aptamer and the Golgi apparatus within HeLa cells. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0042] Unless otherwise specified, the experimental methods described in the following examples are performed using conventional methods and conditions or by referring to the instructions for commercial reagents. Unless otherwise specified, all reagents and consumables involved in this invention are commercially available products.

[0043] Example 1. Design of random ssDNA library, FC-modified capture sequence and PCR primers The design consists of an 80 nt ssDNA library, which comprises a 40 nt random sequence in the center and a 20 nt constant sequence on the flanks: 5'-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3' (SEQ ID NO:8), where N is any one of the four deoxyribonucleotides A, T, C, and G, and 40 represents the number of random nucleotides.

[0044] A fluorocarbon chain-modified capture sequence, FC-CS, with the sequence 5'-TTCACGGTAGCACGCATAGG-3' (SEQ ID NO: 1), was designed to be perfectly complementary to the constant regions of the ssDNA library. Multiple fluorocarbon chain molecules were precisely ligated to the 5' end of the DNA sequence using a phosphorusamide chemistry method, such as... Figure 1 As shown in A, in this embodiment, a total of 9 fluorocarbon chains (CF3(CF2)7CH2CH2—) are introduced into FC-CS. To address the electrostatic repulsion between DNA strands caused by the inherent strong electronegativity of fluorine atoms, Mg is introduced into the system. 2+ The isocations are used to neutralize the negative charge, thereby driving and stabilizing the fluorocarbon chain-mediated self-assembly process. To ensure the controlled release of DNA into the cell, a disulfide bond (-SS-) is inserted between the fluorine modification and the DNA sequence. This bond can be triggered by high concentrations of reduced glutathione (GSH) in the cell, causing it to break and releasing the nucleic acid library into the cytoplasm. Specifically, the preparation method of the fluorocarbon chain-modified capture sequence FC-CS is as follows (…). Figure 1 ): Step 1: Synthesis of FC phosphoramide monomer 1H,1H,2H,2H-perfluoro-1-heptadecyl alcohol was dissolved in dry dichloromethane with N,N-diisopropylethylamine and 2-cyanoethyl-N,N-diisopropylphosphochloroamide and stirred at room temperature for 2 hours. After the reaction was completed, 5% sodium bicarbonate aqueous solution was added to terminate the reaction. The organic phases were extracted with dichloromethane and combined. After drying with anhydrous sodium sulfate, the mixture was concentrated under reduced pressure. The residue was co-evaporated with acetonitrile and used directly for subsequent DNA synthesis. Step 2: Solid-phase synthesis of FC-CS A solid-phase phosphorus amide chemical method was used to synthesize DNA at a scale of 1.0 μmol on an automated DNA synthesizer. Disulfide groups were introduced sequentially at the ends of the DNA, and two three-armed phosphorus amide branching units were coupled to increase the number of linking sites to 9. The DNA was then coupled with FC phosphorus amide monomers to form DNA modified with 9 fluorocarbon chains. Except for the extended coupling time of the fluorocarbon chains and branching monomers to 15 minutes, the rest of the synthesis steps were completed according to conventional operation. Step 3: Cleavage, deprotection, purification, and identification of FC-CS After synthesis, the oligonucleotides were cleaved from the solid-phase support, and the protecting groups were removed by overnight incubation in ammonia solution at room temperature in the dark. The purified products were then purified by reversed-phase high-performance liquid chromatography using a linear gradient of 100 mM triethylamine acetate-acetonitrile. The obtained products were identified by liquid chromatography-mass spectrometry in negative ion mode, and the measured molecular weight was consistent with the theoretical value.

[0045] The upstream PCR primer was modified with Cy5 fluorescence (Cy5-F), with the sequence 5'-AGCAGCACAGAGGTCAGATG-3' (SEQ ID NO: 2), and the downstream primer was modified with biotin (Biotin-R), with the sequence 5'-TTCACGGTAGCACGCATAGG-3' (SEQ ID NO: 3). The fluorescent tag facilitates gel electrophoresis analysis and flow cytometry detection during the screening process; the biotin modification facilitates the separation and purification of the amplified dsDNA product using streptavidin magnetic beads and the preparation of ssDNA for use as the next round of screening library.

[0046] Example 2. Preparation, characterization, and optimization of cell delivery parameters of fluorocarbon chain-nucleic acid library nanoparticles The ssDNA library and the fluorocarbon chain-modified capture sequence FC-CS were mixed at a 1:1 molar ratio in DMEM basal medium containing 10 mg / mL MgCl₂ and incubated at 37°C or 25°C for 15 min, 30 min, 45 min, and 60 min, respectively, to allow the library to be loaded and self-assembled into nanoparticles. Figure 1 (B in the text); non-denaturing polyacrylamide gel electrophoresis confirmed that the assembly products under the above incubation conditions contained no remaining free ssDNA. Figure 2(A) The simplest and quickest incubation condition, "25℃, 15 min", was selected as the subsequent experimental parameter, and the size and monodispersity of the assembled nanoparticles under this condition were characterized using transmission electron microscopy. Figure 2 (B) The cytotoxicity of fluorocarbon chain self-assembled nanoparticles to HeLa cells was evaluated using the CCK-8 assay. After treatment with 10 nM–10 μM particles for 24 hours, cell viability showed no significant decrease compared to the control group. These results indicate that the nanoparticles exhibited no significant cytotoxicity within the tested concentration range, demonstrated good biocompatibility, and are suitable for subsequent intracellular experiments. Figure 2 (C in the middle).

[0047] Before formally conducting in-cell SELEX screening, it is necessary to investigate the fluorocarbon chain-mediated library delivery time window in the HeLa cell system, involving its endocytosis efficiency and endosome / lysosome escape kinetics. By optimizing the incubation time of library nanoparticles with cells, the library can achieve both the highest degree of cell internalization and the lowest degree of endosome / lysosome retention. Flow cytometry and confocal microscopy experiments were used to characterize the endocytosis and subcellular localization of the 5' Cy5 fluorescently modified library.

[0048] HeLa cells cultured in 96-well plates were removed from the incubator, the culture medium was discarded, and the cells were washed twice with PBS buffer. The prepared library nanoparticles were prepared in DMEM basal medium containing 10 mM MgCl2 and added to the wells of the plates. The incubation volume was 50 μL, the final library concentration was 1 μM, and the cells were incubated at 37°C in the dark. Experimental groups (n=3) were set up with untreated cells and incubation times of 1 h, 2 h, 4 h, and 6 h. The samples treated in the experimental groups included assembled fluorescently modified library nanoparticles (FC×Lib) and an equal volume of unassembled fluorescently modified library (free Lib). After incubation, the solution containing the samples was discarded, and the cells were gently washed twice with pre-cooled PBS buffer containing 500 mM NaCl while in an adherent state to remove DNA attached to the cell membrane to avoid fluorescence interference. The cells were then rinsed once more with PBS buffer. After trypsin digestion, the digestion was terminated with complete culture medium, centrifuged, collected, and resuspended in PBS before being analyzed by flow cytometry. The mean fluorescence intensity (MFI) of cells in each group was analyzed to assess DNA endocytosis efficiency. For example... Figure 3 As shown in Figure A, the fluorescence intensity of the experimental group cells increased with the extension of incubation time; under the incubation time gradient of 1 to 6 h, the FC×Lib group showed significantly enhanced fluorescence intensity compared with the free Lib group, indicating that the fluorocarbon chain-mediated self-assembled nanoparticles can effectively enhance the endocytosis of the library.

[0049] HeLa cells cultured in confocal microscopy dishes were removed from the incubator, the culture medium was discarded, and the cells were washed twice with PBS buffer. The assembled FC×Lib nanoparticles were added to the culture dish, and the incubation volume was 100 μL (covering the glass bottom). The final library concentration was 1 μM, and the cells were incubated at 37°C in the dark. Experimental groups were set up with incubation times of 1 h, 2 h, 4 h, and 6 h. After incubation, the solution containing the sample was discarded, and the cells were gently washed twice with pre-cooled PBS buffer containing 500 mM NaCl while in an adherent state to remove DNA attached to the cell membrane surface to avoid fluorescence interference. The cells were then rinsed once more with PBS buffer. The cell nuclei and lysosomes were stained with commercial dyes Hoechst 33342 and LysoTracker Green, respectively. Confocal microscopy was then used to analyze the fluorescence intensity of the Cy5-labeled library in the cells and its Pearson correlation (PCC) with the green fluorescence of lysosomes. With prolonged incubation time, the fluorescence intensity of the intracellular library gradually increased, consistent with the results of flow cytometry experiments. PCC showed a trend of first increasing and then decreasing: 0.63 in the 1 h group, rising to 0.83 in the 2 h group, decreasing to 0.53 in the 4 h group, and slightly recovering to 0.63 in the 6 h group. This kinetic distribution indicates that FC×Lib nanoparticles rapidly accumulate in the acidic compartment during the early endocytosis phase (1–2 h); after 2 h, a large number of nanoparticles successfully escaped the lysosomal pathway, leading to a sharp decrease in PCC; however, in the subsequent time, due to the continuous increase in endocytosis and the balance of intracellular dynamic transport, PCC tends to stabilize. This result strongly demonstrates that this nanomaterial possesses significant endosome / lysosomal escape capability, with the escape window mainly occurring between 2 and 4 h after endocytosis. Figure 3 (B in the original text). Simultaneously, the internalization streaming MFI data under time gradients were normalized, and the ratio of the internalization coefficient to the PCC (E / P) was further calculated. The E / P ratio reached its maximum value in the 4-h group and showed no significant difference from the 6-h group. Figure 3 (C in the text). Therefore, an incubation time of 4–6 h is the optimal incubation time to achieve both cell internalization and endosome / lysosome escape.

[0050] Subsequently, the dopamine cytokinesis of the library delivery needs to be investigated to determine the initial library input amount and its relationship with the cell number during the first round of screening. This is to ensure maximum screening efficiency, avoid false negative or false positive results due to inappropriate dosage, and consider costs to avoid library waste caused by excessive dosage. HeLa cells (1×10⁻⁶ cells / well) were cultured in 48-well plates. 5 / well), set up a library dosage gradient (0-80 pmol), and incubate the assembled fluorocarbon chain-library nanoparticles with cells at 37°C in the dark for 4 h with the corresponding gradient dosage. After incubation, discard the solution containing the sample, and gently wash the cells twice with pre-cooled PBS buffer containing 500 mM NaCl while they are in adherent condition to remove DNA attached to the cell membrane to avoid fluorescence interference. Then wash once more with PBS buffer. After trypsin digestion, the cells were terminated with complete culture medium, centrifuged, collected, and resuspended in PBS for immediate analysis. Analyze the relationship between the MFI of Cy5 fluorescence and the library dosage in each group of cells, and fit the data to a four-parameter logistic equation: Y = Bottom + (Top - Bottom) / (1 + 10^((LogEC50 - X)) HillSlope)). In 1×10 5 In a cellular system, the endocytosis dose-response curve of HeLa cells to fluorocarbon chain-library nanoparticles is as follows: Figure 3 As shown in D, the EC50 is 12.8 pmol and the EC90 is 31.3 pmol (R² = 0.99). Therefore, it can be inferred that at 1 × 10⁻⁶ pmol... 7 The initial library loading dose for In-cell SELEX in the cell system should be in the range of 1280–3130 pmol.

[0051] In summary, to ensure the high efficiency and reliability of In-cell SELEX screening, it is necessary to elucidate the key parameters of fluorocarbon chain-mediated library self-assembly nanoparticles in cell delivery. An incubation time of 4–6 h is optimal for achieving both cell internalization and endosome / lysosome escape, and the initial library dosage should be between 1280 and 3130 pmol (1 × 10⁻⁶ pmol). 7 When the cell system is within a certain range, it is beneficial to ensure the maximization of screening efficiency.

[0052] Example 3. Screening of mitochondrial-targeted intracellular in situ nucleic acid aptamers In-cell SELEX screening is performed in 1×10-1 rounds. 7The experiment was conducted in a HeLa cell system. HeLa cells cultured in 10 cm dishes were removed from the incubator, the culture medium was discarded, and the cells were washed twice with PBS buffer. The assembled library nanoparticles were then added to the culture dish at a final library concentration of 1 μM, and incubated at 37°C to allow for full internalization. For the first round of screening, 2540 pmol (2 OD) of ssDNA library was used for fluorocarbon chain-mediated assembly of the library nanoparticles. Cells were incubated in DMEM containing 10 mM MgCl2 for 5 h. To improve screening efficiency, the screening pressure could be increased by reducing the library input and incubation time with each round of screening. For the second and third rounds, 500 pmol of library was added, and cells were incubated for 5 h. For the fourth and fifth rounds, the library input was reduced to 300 pmol, and the incubation time was reduced to 4.5 h and 4 h, respectively.

[0053] After each round of incubation between the library nanoparticles and cells, the cells are gently washed 2-3 times with pre-cooled PBS buffer containing 500 mM NaCl while still in adherent condition to thoroughly remove DNA attached to the cell membrane. This is followed by a final wash with PBS buffer. Cells are then trypsinized, the incubation is terminated with complete culture medium, centrifuged, collected, and resuspended in PBS. After another centrifugation and discarding the supernatant, mitochondria are isolated and purified using a commercially available kit.

[0054] Add preheated 0.5×TE buffer to the purified mitochondria and incubate at 95°C for 5 min for elution. High temperature denatures the ssDNA and causes it to dissociate from the target. Centrifuge at 14000 g for 15 min and collect the supernatant to obtain mitochondrial-targeted ssDNA. Take 1-2 μL of the product and quantitatively elute the ssDNA yield with Nanodrop and calculate the screening recovery rate (mitochondrial eluted ssDNA yield / ssDNA library input in this round) to help monitor the screening process. Dilute the eluted ssDNA 5-fold with enzyme-free water and use it as a template for PCR amplification (ssDNA template). The 100 μL PCR reaction system is as follows: Hot start premix (2×) 50 μL Cy5-F (10 μM) 5 μL Biotin-R (10 μM) 5 μL 2.5 μL ssDNA template ddH2O 37.5 μL Amplification conditions: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 10 s, 20 cycles, 72℃ final extension for 30 s, and temporary storage at 4℃.

[0055] Preparation of single-streptavidin secondary libraries: Take an appropriate amount of streptavidin magnetic beads and wash them twice with PBS buffer; incubate the PCR amplification product and 1 / 5 volume of 4M NaCl with the magnetic beads for 1 h to immobilize dsDNA on the magnetic beads; discard the supernatant and wash the magnetic beads three times with PBST buffer (PBS buffer containing 0.1% Tween-20); add 100 mM NaOH aqueous solution and incubate with the magnetic beads for 2 min to lyse the dsDNA into ssDNA; add 1 / 10 volume of alkali denaturing solution (100 mM NaOH aqueous solution) of 1 M Tris-HCl (pH 8.0) to a new EP tube in advance, collect the alkali denaturing supernatant into the premixed tube, and mix immediately; then add an equal volume of 2×TE buffer to the liquid in the tube, and use or store at -20℃.

[0056] In summary, the In-cell SELEX screening process mainly includes: library nanoparticle assembly, cell incubation and endocytosis, organelle isolation and purification, target-binding sequence elution and amplification, and secondary library preparation. Figure 4 The above key steps are repeated cyclically to enrich the dominant sequences in the initial random library, which gradually evolve into mitochondrial-targeting nucleic acid aptamers.

[0057] Example 4. Sequencing of nucleic acid aptamer sequence pool and performance characterization of candidate sequences The PCR products obtained from the fifth round of screening were subjected to TA cloning and sequencing, and 50 clones were randomly selected to obtain sequence information. Multiple sequence alignment of the sequencing results was performed using Clustal Omega. All sequences were 80 nt in length and the constant sequences were consistent with the initial library, without truncation, elongation, or mutation. Four sets of repetitive sequences were found among the 50 sequencing results. The first sequence group appears 3 times, and its sequence information is: 5'-AGCAGCACAGAGGTCAGATGGTCACCTTGATCCTAGACACGCGCTGACTAAACCCCTGACCCTATGCGTGCTACCGTGAA-3' (SEQ ID NO: 4), named MitoApt-08; the other 3 repeating sequences each have a repeating frequency of 2 times, and their names and sequences are as follows: MitoApt-46: 5'-AGCAGCACAGAGGTCAGATGACGGATCTTGCGCCGTTCATACACCCTTCGTCCCCCTTCGCCTATGCGTGCTACCGTGAA-3' (SEQ ID NO: 5), MitoApt-77: 5'-AGCAGCACAGAGGTCAGATGACCGTTTCTTAGCGTCACATTTGTCTCTCTATGGTCTCACCCTATGCGTGCTACCGTGAA-3' (SEQ ID NO: 4). 6) and MitoApt-78: 5'-AGCAGCACAGAGGTCAGATGTGGAAGCCCGCGCTGAAGAACGCTGCCAAGACGGGTAGTCCCTATGCGTGCTACCGTGAA-3' (SEQ ID NO: 7). These repetitive sequences were specifically enriched during SELEX iterations, gradually evolving into dominant sequences from the original large-capacity random library. The sequences with the highest frequency and relatively high occurrence represented the most stable or optimal candidate aptamers for target binding and were preferentially selected for synthesis and performance characterization.

[0058] The four aptamers with Cy5 fluorescence modification at 5' were chemically synthesized and mixed with FC-CS at a 1:1 molar ratio in DMEM basal medium containing 10 mM MgCl2. They were incubated at 25°C for 15 min to allow self-assembly into nanoparticles. The subcellular localization of the candidate aptamers in HeLa cells was imaged using confocal scanning microscopy. 100 μL of nanoparticles with a final concentration of 1 μM of candidate aptamers were incubated with cells at 37°C in the dark for 4 h (the preferred time determined in Example 2). After incubation and cell washing, the cells were stained with MitoTracker Green, LysoTracker Green, GolgiTracker Green, and Hoechst 33342, respectively. Subcellular localization of the aptamers (red fluorescence) relative to mitochondria, lysosomes, Golgi apparatus (green fluorescence), and nucleus (blue fluorescence) was then imaged, and the PCC coefficients of the aptamers and organelles were analyzed. Figure 5 As shown, all candidate aptamers exhibited good and specific mitochondrial co-localization. The PCC coefficients of the aptamer's red fluorescence and the mitochondrial's green fluorescence ranged from 0.92 to 0.96, representing a significant improvement in mitochondrial localization compared to the initial library, indicating that their targeting performance gradually evolved with iteration. Furthermore, Figure 6 and Figure 7 The co-localization of the aptamers with lysosomes and the Golgi apparatus within the cell was demonstrated. All candidate aptamers exhibited significantly lower PCC coefficients compared to the Library, indicating aptamer specificity. These results demonstrate that In-cell SELEX yielded mitochondrial-targeting aptamers that specifically localize to mitochondria while exhibiting extremely low co-localization with non-target organelles.

[0059] The above results demonstrate that, relying on in situ intracellular nucleic acid aptamer screening technology, this invention has successfully screened and obtained specific nucleic acid aptamers targeting mitochondria in HeLa cells, providing a reserve for the development of molecular tools targeting subcellular structures and their application in precision medicine.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A fluorocarbon chain-mediated intracellular in situ screening method for organelle-targeting nucleic acid aptamers, characterized in that, Includes the following steps: An ssDNA library was loaded with a fluorocarbon chain-modified capture sequence and self-assembled into nanoparticles. These nanoparticles were then incubated with cells for intracellular delivery. After washing the cells to remove surface-attached nucleic acids, the target organelles were isolated and purified. The ssDNA attached to the target organelles was eluted and used as a template for PCR amplification. The PCR-amplified dsDNA product was prepared into ssDNA for the next round of library screening. The above steps were repeated multiple times to gradually enrich the dominant sequences and identify candidate nucleic acid aptamers through cloning and sequencing. The ssDNA library consists of a 40 nt random sequence in the middle and a 20 nt constant sequence on the flanks, as shown in SEQ ID NO: 8; The fluorocarbon chain-modified capture sequence is a fluorocarbon chain-modified capture sequence FC-CS that is completely complementary to the constant sequence of the ssDNA library. The base sequence of the capture sequence FC-CS is shown in SEQ ID NO: 1, and its 5' or 3' end is modified with 6-12 fluorocarbon chains, with disulfide bonds embedded between the fluorocarbon chains and the base sequence; the fluorocarbon chains are selected from C 10 F 21 CH2CH2-、C8F 17 CH2CH2-, and C6F 13 At least one of CH2CH2-.

2. The method according to claim 1, characterized in that: in, The cells in question are mammalian cells.

3. The method according to claim 2, characterized in that: in, The mammalian cells mentioned are tumor cells, immune cells, nerve cells, epithelial cells, or mesenchymal cells.

4. The method according to claim 3, characterized in that: wherein, The tumor cells are HeLa cells, A549 cells, MCF-7 cells, HCT116 cells, or Jurkat cells.

5. The method according to claim 1, characterized in that: in, The target organelle is a specific subcellular structure or organelle of the cell.

6. The method according to claim 5, characterized in that: wherein, The specific subcellular structures or organelles are organelles related to energy metabolism, protein synthesis and processing, material degradation, genetic material storage and processing, or regions related to cell signal transduction.

7. The method according to claim 1, characterized in that: wherein, The primer pair used for the PCR amplification is the upstream primer shown in SEQ ID NO: 2 and the downstream primer shown in SEQ ID NO:

3. The upstream primer is modified with a fluorescent group, and the downstream primer is modified with biotin.

8. A reagent or kit, characterized in that: The reagent or kit is used to screen nucleic acid aptamers according to any one of claims 1-7, wherein the reagent or kit comprises an ssDNA library and a fluorocarbon chain-modified capture sequence; wherein: The ssDNA library consists of a 40 nt random sequence in the middle and a 20 nt constant sequence on the flanks, as shown in SEQ ID NO: 8; The fluorocarbon chain-modified capture sequence is a fluorocarbon chain-modified capture sequence FC-CS that is completely complementary to the constant sequence of the ssDNA library. The base sequence of the capture sequence FC-CS is shown in SEQ ID NO: 1, and its 5' or 3' end is modified with 6-12 fluorocarbon chains, with disulfide bonds embedded between the fluorocarbon chains and the base sequence; the fluorocarbon chains are selected from C 10 F 21 CH2CH2-、C8F 17 CH2CH2-, and C6F 13 At least one of CH2CH2-.

9. The reagent or kit according to claim 8, characterized in that: The reagent or kit further includes a PCR amplification primer pair, wherein the primer pair is an upstream primer with the sequence shown in SEQ ID NO: 2 and a downstream primer with the sequence shown in SEQ ID NO: 3, wherein the upstream primer is modified with a fluorescent group and the downstream primer is modified with biotin.

10. A mitochondrial-targeting nucleic acid aptamer screened by the method of any one of claims 1-7, characterized in that: in, The sequence of the aptamer is shown in any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO:

7.

11. The mitochondrial-targeting nucleic acid aptamer according to claim 10, characterized in that: in, The 5' or 3' of the aptamer contains at least one chemical modification that is not naturally occurring.

12. The mitochondrial-targeting nucleic acid aptamer according to claim 11, characterized in that: in, The chemical modification is selected from one or more of the following: phosphate group, amino group, thiol group, biotin, digoxigenin, fluorescent labeling, quenching group, lipid molecule, polyethylene glycol molecule, and nucleotide analog.

13. The application of the mitochondrial-targeting nucleic acid aptamer according to claim 10, characterized in that, The applications are selected from the following group: i) applications in the preparation of subcellular imaging probes; ii) applications in the preparation of mitochondrial targeted delivery tools.

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