An intracellular imaging system for simultaneously detecting UDG and miR-375 expression levels and its application

Abstract

This invention discloses an intracellular imaging system for simultaneously detecting UDG and miR-375 expression levels and its applications. The intracellular imaging system uses a tetrahedral DNA nanostructure as a carrier, loading a HAT-UDG probe with an on / off conformation, and coupling it with a split CRISPR / Cas12a detection component for recognizing miR-375. The split CRISPR / Cas12a detection component includes at least Cas12a protein, scaffold RNA, spacer RNA / equivalent split crRNA components, and a fluorescent reporter substrate. This invention is the first to perform a logical AND operation between two key indicators: UDG enzyme activity and miR-375 expression level. The system can only ultimately trigger a complete fluorescence signal when both are simultaneously highly expressed. This multi-target synergistic activation design constructs a precise molecular recognition logic gate, enabling extremely accurate differentiation between prostate cancer cells (RV-1) and normal cells (293T), as well as other cancer cells (5637) that highly express UDG, fundamentally avoiding misjudgments caused by fluctuations in a single indicator or non-specific expression, resulting in extremely high imaging specificity.

Description

Technical Field

[0001] This invention belongs to the field of intracellular imaging technology, and particularly relates to an intracellular imaging system for simultaneously detecting the expression levels of UDG and miR-375 and its applications. Background Technology

[0002] Intracellular imaging technology, as a core tool in modern life sciences and medical diagnostics, aims to visualize the dynamic changes of specific biomolecules such as proteins, nucleic acids, enzymes, and ions within living cells. Currently, technological development in this field primarily relies on the following probe systems: gene-encoded fluorescent probes, represented by green fluorescent protein (GFP) and its derivatives, can be fused with target proteins through genetic engineering to achieve real-time monitoring of protein localization, expression, and interactions; probes based on FRET (fluorescence resonance energy transfer) technology can further be used to detect enzyme activity, ion concentration changes, etc. Synthetic small-molecule fluorescent probes are small molecule compounds capable of penetrating cell membranes; their fluorescence properties, such as intensity and wavelength, change upon binding to specific targets, thus enabling detection. Furthermore, functional nucleic acid probes are one of the fastest-growing areas, including molecular beacons for detecting specific mRNAs or miRNAs, nucleic acid aptamer probes that specifically bind to non-nucleic acid targets, and DNA nanomachines designed through ingenious self-assembly that can perform complex tasks within cells, such as walking, computation, and signal amplification, aiming to achieve multiplex, highly sensitive imaging. Meanwhile, nanomaterial probes, such as quantum dots, upconversion nanoparticles, and carbon dots, are widely used as high-performance fluorescent tags for long-term tracking and super-resolution imaging due to their excellent optical properties, including high brightness, resistance to bleaching, and tunable emission wavelengths. Overall, this technological field is undergoing a profound evolution from single-target and endpoint detection to multi-target, real-time dynamic, and even ultra-high-resolution imaging, and is increasingly showing a trend towards "therapeutic integration" that combines diagnosis and treatment.

[0003] Despite significant advancements in intracellular imaging technology, existing methods still face a series of recognized technical bottlenecks and challenges when applied to precision medicine, such as the development of fluorescence navigation systems. First, the biocompatibility and toxicity of probes are prominent issues. Many exogenous probes, especially some synthetic probes and nanomaterials, can non-specifically interfere with normal cellular physiological activities and even induce stress responses, leading to observational distortions. Furthermore, certain materials, such as cadmium-containing quantum dots, can cause metal ion leakage and phototoxicity from light exposure, further damaging cell viability and making long-term imaging difficult. Second, low imaging efficiency and signal-to-noise ratio constitute core obstacles. Specifically, this manifests as low probe targeting efficiency, easy non-specific adsorption leading to high background noise, weak signal output from individual probes resulting in blurred imaging of low-abundance targets, and the "signal leakage" problem commonly found in nucleic acid probes, causing false positive results. Third, existing technologies are insufficient in complex application scenarios. On the one hand, limitations in multiplex detection capabilities, such as crosstalk in the fluorescence spectra of different probes and difficulties in simultaneous and efficient delivery, hinder the simultaneous and accurate imaging of multiple targets. On the other hand, most probes lack dynamic regulation capabilities; their irreversible activation modes cannot be actively controlled by "on / off," making it difficult to study dynamic biological processes. Finally, and most importantly, their insufficient cell selectivity (specificity) restricts their clinical translation. Existing probes typically cannot accurately distinguish and specifically stain target cell types (such as prostate cancer cells) in complex tissue microenvironments, making them unsuitable as reliable "navigation" systems for accurately guiding surgical boundaries or performing in vivo diagnosis, thus greatly limiting their practical application value in fluorescence navigation.

[0004] The problems faced by existing intracellular imaging technologies can be attributed to inherent limitations at three levels: materials, design, and biological environment. At the materials level, the core components of many high-performance nanomaterials (such as quantum dots) are inherently toxic metals, raising questions about their biosafety. Synthetic small-molecule probes, due to their inherent hydrophobicity or specific charges in their chemical structures, are prone to non-specific embedding in biological membranes or undesirable interactions with biomolecules, thereby interfering with normal physiological functions. At the probe design level, there are several conceptual flaws: First, traditional "always-on" designs do not adequately consider background signal suppression, resulting in a naturally low signal-to-noise ratio. Second, probes generally lack effective active targeting modules, relying mainly on passive diffusion after entering the cell, making it difficult to be precisely "navigated" to specific subcellular sites like natural proteins. Third, there is a contradiction between stability and function; for example, chemical modifications to enhance stability often sacrifice target binding efficiency or membrane penetration. Ultimately, all designs must face the challenges of the complex intracellular environment. Probes face multiple obstacles, including enzymatic degradation, vesicle isolation, efflux pump clearance, and competitive binding with non-target molecules. Failure in any of these steps will lead to the failure of the entire imaging process.

[0005] In the research and development process of overcoming the aforementioned technical challenges, those skilled in the art face a variety of intertwined technical difficulties from multiple dimensions. First is the fundamental difficulty of balancing performance and biosafety. The greatest challenge lies in designing a material or molecule that simultaneously achieves excellent optical properties with absolute biocompatibility and non-toxicity. For example, developing heavy metal-free nanomaterials to replace traditional cadmium-containing quantum dots often fails to achieve the same level of fluorescence quantum yield and long-term stability, making it difficult to achieve both performance and safety. Second is the systemic difficulty of achieving efficient and specific delivery. A key challenge is that the targeting groups introduced to improve targeting often increase the molecular weight and structural complexity of the probe, potentially impairing its most basic cell penetration efficiency, creating a design paradox. Third is the inherent difficulty of designing high signal-to-noise ratio probe structures. Constructing probe structures that can maintain extremely low background in complex intracellular environments and generate extremely high signal-to-noise ratio signals at the target site is a core challenge in molecular design. This requires the probe to possess near-perfect "on / off" properties. Furthermore, designing reversible probes capable of real-time monitoring of physiological processes requires the introduction of biocompatible external control switches, such as specific wavelength light sources or specific biomarkers, which are extremely challenging in terms of chemical implementation and integration. Then there is the difficulty of synergistic optimization when integrating multiple detection functions. Achieving simultaneous imaging of multiple intracellular targets presents not only the challenge of resolving spectral crosstalk between different fluorescence channels, but also ensuring that multiple probes targeting different targets do not cross-react during co-delivery and possess matching delivery efficiency, kinetic behavior, and metabolic stability. This requires precise "system-level" synergistic optimization of all components, with the workload and technical complexity increasing exponentially. Finally, there is the significant hurdle of transitioning from in vitro validation to in vivo application. Many probes that perform exceptionally well under simulated conditions or in vitro buffers experience a sharp decline in performance due to various unforeseen interferences once placed in the real intracellular environment. Therefore, endowing probe designs with sufficient "robustness" to tolerate and adapt to the ever-changing real conditions within cells is the ultimate obstacle to realizing their final clinical application value. Summary of the Invention

[0006] The purpose of this invention is to provide an intracellular imaging system and its application for simultaneously detecting the expression levels of UDG and miR-375. Only when both are highly expressed can a complete fluorescence signal be triggered. This multi-target synergistic activation design constructs a precise molecular recognition logic gate, which enables it to distinguish prostate cancer cells (RV-1) from normal cells (293T) and even other cancer cells (5637) that highly express UDG, fundamentally avoiding misjudgment caused by fluctuations or non-specific expression of a single indicator, and achieving extremely high imaging specificity.

[0007] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides an intracellular imaging system for simultaneously detecting the expression levels of UDG and miR-375. The intracellular imaging system uses a tetrahedral DNA nanostructure as a carrier, loads a HAT-UDG probe with a switchable conformation, and couples it with a split CRISPR / Cas12a detection component for recognizing miR-375. The split CRISPR / Cas12a detection component includes at least Cas12a protein, scaffold RNA, spacer RNA / equivalent split crRNA components, and a fluorescent reporter substrate.

[0008] Wherein, the spacer RNA is miR-375; the scaffold RNA contains a binding region that can hybridize with miR-375, so that when miR-375 is present, it assembles with the scaffold RNA to form a crRNA with a guiding function, the crRNA including a scaffold region and a spacer region, thereby activating the Cas12a protein to trans-cleavage the fluorescent reporter substrate and generate a fluorescent signal; the equivalent split crRNA component is an alternative split form that can achieve the above assembly and activation functions.

[0009] The tetrahedral DNA nanostructure (TDN) of this invention is a conventional tetrahedral DNA self-assembly structure in the art, formed by the complementary pairing of four oligonucleotide chains. To achieve targeted probe loading and improved intracellular stability, this invention preferably introduces an epitaxial linker (including a polyT spacer arm and a docking sequence) at the end of the TDN chain, and may further employ phosphorus sulfide modification to enhance resistance to degradation; the above-mentioned linker and modification are preferred embodiments and do not constitute the only limitation of the TDN.

[0010] The HAT-UDG probe of this invention is preferably loaded onto the TDN via sequence complementary hybridization: that is, an epitaxial linker (including a polyT spacer arm and a docking sequence) is set at one or more vertices of the TDN, and the HAT-UDG probe is set with a binding region complementary to the docking sequence. After annealing and hybridization, the two achieve site-specific loading. The loading site is preferably a TDN vertex to obtain a defined spatial configuration and low steric hindrance; without affecting the HAT switching conformation and the release of the single-stranded probe, the loading site can also be set at other predetermined sites on the TDN.

[0011] In one embodiment, the HAT-UDG probe includes a cap-shaped / bubble-shaped structure region formed by polyT, and introduces at least one deoxyuridine as an UDG recognition site in the cap-shaped / bubble-shaped structure region.

[0012] In one embodiment, the HAT-UDG probe is loaded onto a predetermined site on the vector via sequence complementary hybridization or coupling.

[0013] As one embodiment, the HAT-UDG is loaded onto an extensional connection sequence of one or more vertices of the carrier.

[0014] As one embodiment, the epitaxial connection sequence includes a polyT spacer arm and a docking sequence.

[0015] As one embodiment, the nucleotide sequence of the HAT-UDG probe is: 5′-TGGAGAGGGCG-(T)10-dU-(T)10-GCCAGGGGTATGTG-3′.

[0016] The dU site is located within the polyT cap / bubble-like structure region of the HAT probe and serves as the substrate recognition site for UDG. In one embodiment, the dU modification site of HAT-UDG-PROBE is located at the center of the probe's polyT bubble-like region: that is, 10 Ts are linked after the 11th base at the 5′ end, followed by the introduction of one dU (at position 22 of the sequence), and then another 10 Ts are linked, thereby forming an enzymatically responsive open-pore structure of (T)10-dU-(T)10. After UDG recognizes and removes the dU, an AP site is formed at this site. APE1 then cleaves at the AP site, causing the HAT probe to break / unblock at this location, thereby removing steric hindrance and releasing (or exposing) the single-stranded output segment required for subsequent reactions, achieving a controllable switch from "off" to "on".

[0017] Secondly, the present invention provides the application of the above-mentioned intracellular imaging system in the diagnosis of prostate cancer.

[0018] Thirdly, the present invention provides the application of the above-mentioned intracellular imaging system in defining the surgical boundaries of prostate cancer.

[0019] Fourthly, the present invention provides the application of the above-mentioned intracellular imaging system in the evaluation of the therapeutic efficacy of prostate cancer treatment.

[0020] Fifthly, the present invention provides the application of the above-mentioned intracellular imaging system in the screening of candidate drugs for prostate cancer.

[0021] In addition, it should be noted that the intracellular imaging system of the present invention can also be applied to: (1) Risk stratification / grading of prostate cancer (2) Application in recurrence risk prediction / prognostic assessment (e.g., postoperative recurrence or metastasis risk assessment) (3) Metastasis-related imaging / micrometastasis cell recognition (4) Imaging analysis after identification / enrichment of circulating tumor cells (CTCs) (5) Assessment of treatment sensitivity / resistance (e.g., ADT, AR pathway inhibitors, chemotherapy or radiotherapy sensitivity).

[0022] (6) Treatment plan selection / individualized medication guidance (7) Research on the activity of DNA damage repair-related pathways or miRNA regulatory mechanisms (8) Intracellular imaging analysis of prostate tissue sections / biopsy samples / intraoperative rapid pathological samples (9) Organoid / Patient-Derived Cell (PDC) / Patient-Derived Xenograft (PDX) Cell Models (10) Intracellular imaging and diagnosis of solid tumors, including but not limited to prostate cancer.

[0023] The beneficial effects of this invention are as follows: (1) Highly specific multi-target co-activation imaging was achieved, significantly improving accuracy. Existing technologies often rely on single biomarkers for detection, which can easily lead to false positives in the complex tumor microenvironment. This invention, for the first time, uses a logical AND operation between two key indicators: UDG enzyme activity and miR-375 expression level. The system can only ultimately trigger a complete fluorescence signal when both are simultaneously highly expressed. This multi-target synergistic activation design constructs a sophisticated molecular recognition logic gate, enabling it to distinguish prostate cancer cells (RV-1) from normal cells (293T) and even other cancer cells that highly express UDG (5637) with extremely high precision. This fundamentally avoids misjudgments caused by fluctuations or non-specific expression of a single indicator, resulting in extremely high imaging specificity.

[0024] (2) Innovatively integrate multiple advanced technology platforms to achieve performance breakthroughs. This invention is not a simple superposition of existing technologies, but rather an integrated design that combines a tetrahedral DNA nanostructure, a HAT probe, and a CRISPR-Cas12a system. TDN, acting as a carrier, not only protects the probe but also improves cell delivery efficiency; the HAT probe is responsible for responding to the UDG / APE1 pathway; and the Split Cas12a system serves as a high-fidelity miR-375 sensor. This modular, functionally integrated design achieves a synergistic effect greater than the sum of its parts (1+1+1>3), solving the problem of traditional probes having limited functionality and being unable to work collaboratively in complex pathways.

[0025] (3) It has excellent biocompatibility and low cytotoxicity, ensuring application safety. Experimental data directly demonstrate that the entire system, including the electroporated TDP delivery, has minimal impact on cell viability, with a mortality rate below 5% and no significant difference compared to the control group. This is attributed to the excellent biocompatibility of TDN itself and the minimization of interference with normal cellular physiology after probe encapsulation. Compared to traditional imaging materials based on heavy metal quantum dots, this invention fundamentally eliminates the risk of inherent material toxicity, providing an important safety foundation for future clinical translation.

[0026] (4) It solves the industry pain points of low signal-to-noise ratio and poor stability in complex intracellular environments. Traditional "always-on" probes or single-activation probes are prone to generating high background noise within cells. This invention, through a dual control mechanism of "cascaded amplification" and "split Cas12a activation," ensures extremely low background signal and a very high signal-to-noise ratio. Simultaneously, the structurally stable TDN carrier effectively protects the probe, enabling it to resist degradation by intracellular nucleases, significantly improving the stability and reliability of the probe in the complex intracellular environment, and ensuring clear and accurate imaging signals.

[0027] (5) It provides a new, highly specific tool for the precise diagnosis and fluorescence navigation of cancer. The successful development of this invention goes beyond the scope of "mere detection," achieving "precise identification" of specific cell types. This high-precision cell differentiation capability based on molecular characteristics gives it enormous application potential in the early diagnosis of prostate cancer, surgical boundary delineation (fluorescence navigation), and efficacy evaluation. Attached Figure Description

[0028] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 A schematic diagram illustrating the process by which a HAT open-pore probe (TDP) loaded with a tetrahedral DNA nanostructure enters a cell via electroporation, and is then triggered by the intracellular UDG / APE1 cascade to unblock / release the probe, further activating the split CRISPR / Cas12a system to generate a fluorescent signal.

[0029] Figure 2 A schematic diagram of the mechanism by which UDG and APE1 cascade and lyse HAT open-hole probes in vitro and generate fluorescence signals, and experimental verification results.

[0030] Figure 3 Gel electrophoresis analysis of stepwise assembly of tetrahedral DNA nanostructures (TDN) (corresponding to single-stranded, double-stranded, triple-stranded and quadruple-stranded assembly products, respectively).

[0031] Figure 4 Gel electrophoresis verification diagram of TDN, functionalized TDN (TDN with linker only), and tetrahedral DNA probe (TDP, TDN with linker and probe loaded).

[0032] Figure 5 Bar chart showing quantitative analysis of cell fluorescence signals before and after electroporation delivery.

[0033] Figure 6 : Results of fluorescence activity staining for cell survival / death before and after electroporation delivery (NC: negative control; EP: electroporation group).

[0034] Figure 7 Figure: Results of qPCR detection of miR-375 expression levels in different prostate cancer cell lines.

[0035] Figure 8 Based on publicly available database information, this paper presents a comparative analysis of the immunohistochemical (IHC) results of prostate tissues from patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) (showing representative stained images and / or corresponding quantitative scoring results).

[0036] Figure 9 Image of DNA tetrahedral probes in normal cells and prostate cancer cells using confocal laser scanning microscopy (CLSM). Detailed Implementation

[0037] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0038] This invention provides an intracellular imaging system for simultaneously detecting the expression levels of UDG and miR-375. It uses a tetrahedral DNA nanostructure (TDN) as a carrier to load a HAT-UDG probe with a switchable conformation and couples it with a split CRISPR / Cas12a detection component for recognizing miR-375.

[0039] The split CRISPR / Cas12a detection component includes at least the Cas12a protein, scaffold RNA, spacer RNA (or an equivalent split crRNA component), and a fluorescent reporter substrate for outputting a fluorescent signal upon target triggering. The spacer RNA is miR-375. The scaffold RNA contains a binding region that hybridizes with miR-375, allowing it to assemble with the scaffold RNA in the presence of miR-375 to form a guiding crRNA. The crRNA includes a scaffold region and a spacer region, thereby activating the Cas12a protein to trans-cleavage the fluorescent reporter substrate and generate a fluorescent signal. An equivalent split crRNA component refers to an alternative splitting method that achieves the above assembly and activation functions. Equivalent split crRNA components include, but are not limited to, providing the scaffold region and spacer region of the crRNA as two or more nucleic acid fragments, wherein the spacer region is provided solely by miR-375, or assembled from miR-375 and an auxiliary nucleic acid fragment (DNA-375).

[0040] The HAT-UDG probe contains a cap-like / vesicle-like structure region formed by polyT, and introduces at least one deoxyuridine (dU) as a UDG recognition site at a predetermined location. When the probe is intact, its spatial conformation creates a steric hindrance effect, causing the relevant output strand to be in a closed state or an ineffectively recognized state, thereby suppressing the background signal. The HAT-UDG probe can be loaded onto the predetermined site of TDN via sequence complementary hybridization or coupling, preferably onto an extensional linker at one or more vertices to obtain a well-defined spatial configuration and high stability.

[0041] Intracellularly, when UDG is sufficiently active, it removes dU to form a base-deficient site, which is then cleaved by APE1, disrupting the HAT conformation and removing steric hindrance, thereby releasing or exposing the single-stranded output / trigger sequence for downstream reactions. When the miR-375 content reaches a threshold, miR-375 specifically binds to the recognition region in the split CRISPR / Cas12a component, triggering / stabilizing the activated conformation of Cas12a and generating trans-cleavage. Cas12a significantly cleaves the fluorescent reporter substrate, outputting a complete and detectable fluorescence imaging signal, only when both events occur simultaneously. When either event is absent, the system maintains low background or does not produce significant fluorescence output, thus achieving logical AND determination.

[0042] The preferred methods for constructing and using the intracellular imaging system of this invention include: TDN self-assembly to form a vector; site-specific loading of the HAT-UDG probe onto the TDN to obtain a complex; introducing the complex and split CRISPR / Cas12a-related components into the target cells (electroporation or other delivery methods may be used); acquiring fluorescence signals under a fluorescence microscope or high-content imaging device, and distinguishing between prostate cancer cells and non-target cells based on signal intensity / distribution, as well as applications such as prostate cancer diagnosis, intraoperative boundary delineation, and efficacy evaluation.

[0043] The raw materials for this invention are sourced from: All oligonucleotide chains used in the experiments were synthesized and purified by Sangon Biotech Ltd. (Shanghai, China).

[0044] The concentration of DNA oligonucleotides was measured using a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

[0045] The sequences of all oligonucleotides are shown in Table 1.

[0046] Table 1 Oligonucleotide Sequences

[0047] In the table, * indicates the introduction of phosphorus sulfide modification to improve the stability of the tetrahedron.

[0048] AsCas12a, FnCas12a and LbCas12a were purchased from Tolo Biotech Co., Ltd. (Shanghai, China).

[0049] 2 mM MgSO4 was purchased from New England Biolabs Inc. (Beijing, China).

[0050] Agarose, 5×TBE buffer, 4S Red Plus Nucleic Acid Stain, 6× DNA loading dye, DNA Marker and ATP were purchased from Sangon Biotech Ltd. (Shanghai, China).

[0051] The MiPure Cell / Tissue miRNA Kit, miRNA 1st Strand cDNA Synthesis Kit (bystem-loop), and miRNA Unimodal SYBR qPCR Master Mix were purchased from Vazyme (Jiangsu, China).

[0052] Six cell lines were used in this study, all obtained from BOSTER (Wuhan, China).

[0053] Three prostate cancer cell lines, 22RV1, C42, and LNCAP, bladder cancer cell line 5637, and the common tool cell line 293T were all cultured using RPMI-1640 medium supplemented with penicillin G (100 U / mL), streptomycin (100 μg / mL), and 10% fetal bovine serum (FBS).

[0054] Human normal prostate cell line RWPE1 was cultured in a special medium (K-SFM + 0.05 mg / ml BPE + 5 ng / ml EGF + PS, IMMOCELL, Xiamen, China).

[0055] All cells were placed in a 37°C cell culture incubator with 5% CO2.

[0056] Product process of this invention: (1) Preparation of HAT probe: The HAT probe consists of a main strand of HAT DNA and a reporter strand of HAT-Probe. The nucleotide sequence of the main strand of HAT DNA is shown in SEQ ID NO. 1: TGGAGAGGGCGTTTTTTTTTTTTTTTTTTTTGCCAGGGGTATGTG. The nucleotide sequence of the reporter strand of HAT-Probe is shown in SEQ ID NO. 2: CACATACCCCTGGCCGCCCTCTCCA.

[0057] Take 2 μL of main strand HAT DNA (100 nM), 2 μL of reporter strand HAT-Probe (100 nM), and 14 μL of DEPC water, and mix with 2 μL of 10× r2.1 buffer. Vortex for 1 minute to ensure thorough mixing. Place the mixture in a PCR instrument and anneal according to the following program: 85℃ for 5 minutes, 45℃ for 10 minutes, and 25℃ for 10 minutes. The HAT probe is then successfully prepared.

[0058] (2) Gel electrophoresis to verify the tetrahedral assembly of nucleic acid: The tetrahedral DNA nanostructure (TDN) is formed by the self-assembly of four oligonucleotide backbone chains S1, S2, S3, and S4 according to the design complementary pairing; In order to achieve subsequent probe site loading and stability improvement, the corresponding backbone chains can be replaced with modified chains S1-linker, S2-linker, S3-linker, and S4-linker with extensional linkers. The linker preferably includes a polyT spacer arm and a docking sequence, and **phosphorothioate (marked with "*" in the sequence)** can be introduced at the end of the docking sequence to enhance the resistance to nuclease degradation and improve loading stability; The sequences of the backbone chains and modified chains are detailed in Table 1. Add 4S Red Plus dye to a 3% agarose gel and perform agarose gel electrophoresis at 110 V in 1×TBE buffer. Take 10 µL of sample, mix it with 2 µL of 6× loading buffer, and load the sample onto the gel. Visualize the sample using the Bio-Rad Universal Hood II gel imaging system (Bio-Rad, Shanghai, China).

[0059] (3) Intracellular imaging: 22RV1 cells were placed in a six-well plate at a density of approximately 1 × 10⁻⁶ cells / well. 6 Cells were cultured at a density of [number] cells / well, and after necessary experimental treatments, were cultured at 37°C with 5% CO2 for 24 hours. Subsequently, the cells were stained with Calecein-AM / PI dye (Beyotime Biotech, Shanghai, China) for 10 minutes. Calecein-AM dye showed live cells as green, and PI dye showed dead cells as red. Cells were visualized using a fluorescence microscope (Bio-Rad, USA). Fluorescence intensity was measured on the visualized fluorescence images using FiJi software (https: / / imagej.net / software / fiji / ).

[0060] This invention successfully constructs an intracellular imaging system using tetrahedral DNA nanostructures (TDNs) as carriers for the simultaneous detection of uracil DNA glycosidase (UDG) and miR-375 expression levels. The system loads a deoxyuridine-modified HAT probe onto a nucleic acid tetrahedral TDN, forming a TDP composite probe, which is then delivered into cells via electroporation in conjunction with a Split Cas12a system. In a cellular environment with high UDG and APE1 expression (UDG is responsible for generating AP sites, and APE1 is responsible for cleaving DNA at AP sites; the two are connected in series to form a cascade switch, and only through their continuous action can probe unsealing and release of the output strand be achieved), the dU sites are sequentially recognized and cleaved, releasing single-stranded probes. If high miR-375 expression is also present in the cell, the Split Cas12a system can be further activated, cleaving the probe and generating a FAM fluorescence signal, ultimately achieving multi-target synergistic activation of intracellular fluorescence imaging. (In the cellular environment where UDG and APE1 work synergistically, UDG first specifically recognizes and removes dU from the probe to form an AP site. Subsequently, APE1 recognizes this AP site and cleaves the DNA backbone, thereby disrupting the HAT structure and releasing (or exposing) the single-stranded probe. When miR-375 is simultaneously highly expressed in the cell, the split Cas12a system is further activated and the reporter substrate is trans-cleaved, generating a FAM fluorescence signal, thus achieving intracellular fluorescence imaging with multi-target synergistic activation.) To verify the feasibility of this mechanism, we first conducted in vitro experiments. The results showed that significant fluorescence activation was only observed when the HAT probe, UDG, and APE1 coexisted. The successful assembly of TDN and TDP was further confirmed by gel electrophoresis. In cell experiments, the effect of the electroporation delivery method on cell viability was evaluated. Fluorescence imaging and quantitative analysis showed that the cell death rate in the control group, the electroporation treatment group, and the electroporated TDP-loaded group was all less than 5%, with no significant difference between the groups, demonstrating that this delivery strategy has good biosafety.

[0061] Based on immunohistochemical and miRNA expression data from our database, we selected three cell models: normal cells 293T (low expression of UDG and miR-375), bladder cancer cells 5637 (high expression of UDG and low expression of miR-375), and prostate cancer cells RV-1 (high expression of UDG and miR-375) to examine the cell specificity of the imaging system. Confocal imaging results showed that significant FAM fluorescence signals were detected only in the cytoplasm of RV-1 cells, indicating that the Split CRISPR-Cas12a system was specifically activated only in prostate tumor cells in this model. This result demonstrates that the constructed imaging system possesses excellent cell type discrimination capabilities, laying a reliable foundation for developing highly specific intracellular imaging strategies for tumor cells.

[0062] The core construction strategy of the intracellular imaging system of this invention is to load HAT functional probes modified with deoxyuridine (dU) sites onto the apex of TDN using site-directed coupling technology, forming a structurally stable TDP composite probe. By optimizing electroporation parameters, we efficiently co-deliver the TDP probe and components of the Split Cas12a system into cells. The working principle of this system is based on a cascade recognition and signal amplification mechanism: such as... Figure 1 As shown, in a cellular microenvironment where UDG and APE1 are highly expressed, the dU site is first specifically recognized and cleaved by UDG, followed by cleavage by APE1 at the base-deficient site, thereby releasing the blocked single-stranded probe. When miR-375 is also highly expressed intracellularly, it specifically binds to crDNA in the Split Cas12a system, activating the trans-cleavage activity of the Cas12a protein, thereby cleaving the reporter probe and ultimately generating a strong FAM fluorescence signal. This dual-target synergistic activation design effectively ensures the specificity of the imaging signal, enabling fluorescence imaging for the simultaneous detection of multiple biomarkers at the live-cell level.

[0063] To verify the feasibility of this molecular mechanism, we first conducted a series of verification experiments in vitro, simulating the intracellular environment. The results of fluorescence kinetic monitoring (…) Figure 2 The results showed that significant fluorescence signal enhancement was only observed when the reaction system contained all three components—HAT probe, UDG, and APE1—simultaneously; the control group lacking any of the enzyme components showed no significant signal response. The assembly process of TDN and TDP was systematically characterized by non-denaturing polyacrylamide gel electrophoresis. Figure 3The results showed that the four single chains appeared as diffuse bands in close proximity in the gel. The migration rate of the double-chain mixture was slower than that of the single chains, indicating that a stable double-chain intermediate was formed between the two chains through partial base complementarity. The migration rate of the triple-chain mixture was further slowed, indicating that the three chains formed a more complex triple-chain partial assembly through more complex base complementarity. The electrophoresis results of the quadruple-chain mixture showed a single, compact band, and its migration position was significantly slower than that of any partial assembly, which is consistent with the theoretically predicted molecular weight and three-dimensional size of the complete tetrahedral structure. The uniformity of the bands indicates that the assembly product is homogeneous, with no obvious residual partial assembly byproducts. This suggests that a homogeneous final product can only be formed when the four single chains are completely complementary in a specific ratio and order. Furthermore, as... Figure 4 The assembly process of TDN and TDP was systematically characterized. The results showed that TDN had a characteristic band at about 150 bp, while the TDP complex showed obvious migration lag due to the increase in molecular weight. This confirmed the successful assembly of TDN nanocarrier and HAT probe at the structural level.

[0064] To evaluate the safety of electroporation as a nucleic acid delivery method, this invention systematically analyzed the impact of this process on cell viability. Using a dual fluorescence staining method with calcein-AM (labeling live cells, showing green fluorescence) and propidium iodide (labeling dead cells, showing red fluorescence), we performed staining and imaging analysis on the following three groups of cells: a blank control group without any treatment, a control group that received only electroporation treatment, and an experimental group that was electroporated and loaded with TDP complexes. Figure 5 and Figure 6 As shown in the fluorescence microscopy results, all three groups of cells maintained high cell viability, intact morphology, and good adhesion. One-way ANOVA showed no significant difference in cell mortality among the three groups (P>0.05). These experimental results demonstrate that the electroporation parameters and TDP loading process used had minimal impact on cell viability, and cell mortality was controlled within a biologically acceptable range of less than 5%. This fully demonstrates that the electroporation delivery strategy has good biocompatibility and reliable biosafety, providing important evidence for its further application in cell-level research.

[0065] Database-based immunohistochemical analysis and miRNA expression profiling data ( Figure 7 , Figure 8This invention screened three cell lines with representative UDG and miR-375 expression patterns to verify the cell specificity of the constructed imaging system: human embryonic kidney cells 293T (showing low expression of both UDG and miR-375), bladder cancer cells 5637 (characterized by high UDG expression but low miR-375 expression), and prostate cancer cells RV-1 (showing high expression of both UDG and miR-375). Imaging analysis of the three cell groups was performed using laser confocal microscopy, as shown in... Figure 9 The results showed that no significant FAM fluorescence signal was detected in either 293T or 5637 cells; however, obvious green fluorescence aggregation was observed only in the cytoplasm of RV-1 cells. This phenomenon indicates that the constructed Split CRISPR-Cas12a imaging system can be specifically activated in prostate tumor cells by recognizing the dual signals of UDG and miR-375 within cells, exhibiting excellent cell type resolution capabilities and providing a new technical means for tumor-specific molecular diagnosis.

[0066] This invention's cell imaging system elucidates a complete and ordered molecular chain from "UDG / APE1 cleavage of dU to release single-stranded DNA" to "this single-stranded DNA and miR-375 co-activating Split Cas12a" and then to "Cas12a trans-cleavage of the reporter probe to generate fluorescence." The core of this system's multi-target synergistic activation logic design lies not in simply detecting two targets, but in designing a molecular-level AND gate logic circuit. The system must simultaneously satisfy both "UDG / APE1 restriction pathway activation" and "miR-375 expression" to ultimately generate a fluorescence signal, which greatly improves the specificity and accuracy of imaging.

[0067] The cell imaging system of this invention has been experimentally verified to specifically generate strong fluorescence signals only in prostate cancer cells (RV-1) from a variety of cell types (normal cells, other cancer cells). This proves that it solves the industry pain point of "insufficient specificity" and points out its unique application value in the diagnosis of prostate cancer.

[0068] In addition, the cell imaging system of the present invention takes into account both safety and efficiency. It uses electroporation to deliver the entire system into the cell and has verified that the cell survival rate under this strategy is higher than 95%, which ensures good biosafety while maintaining functionality.

[0069] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An intracellular imaging system for simultaneously detecting the expression levels of UDG and miR-375, characterized in that: The intracellular imaging system uses a tetrahedral DNA nanostructure as a carrier, loads a HAT-UDG probe with a switchable conformation, and is coupled to a split CRISPR / Cas12a detection component for recognizing miR-375. The split CRISPR / Cas12a detection component includes at least Cas12a protein, scaffold RNA, spacer RNA / equivalent split crRNA components, and a fluorescent reporter substrate. Wherein, the spacer RNA is miR-375; the scaffold RNA contains a binding region that can hybridize with miR-375, so that when miR-375 is present, it assembles with the scaffold RNA to form a crRNA with a guiding function, the crRNA including a scaffold region and a spacer region, thereby activating the Cas12a protein to trans-cleavage the fluorescent reporter substrate and generate a fluorescent signal; the equivalent split crRNA component is an alternative split form that can achieve the above assembly and activation functions.

2. The intracellular imaging system according to claim 1, characterized in that: The HAT-UDG probe contains a cap-shaped / bubble-shaped structure region formed by polyT, and introduces at least one deoxyuridine as a UDG recognition site in the cap-shaped / bubble-shaped structure region.

3. The intracellular imaging system according to claim 1, characterized in that: The HAT-UDG probe is loaded into a predetermined site on the vector via sequence complementary hybridization or coupling.

4. The intracellular imaging system according to claim 3, characterized in that: The HAT-UDG is loaded onto an extensional connection sequence of one or more vertices of the carrier.

5. The intracellular imaging system according to claim 4, characterized in that: The epitaxial connection sequence includes a polyT spacer arm and a docking sequence.

6. The intracellular imaging system according to claim 2, characterized in that: The nucleotide sequence of the HAT-UDG probe is: 5′-TGGAGAGGGCG-(T)10-dU-(T)10-GCCAGGGGTATGTG-3′.

7. The application of the intracellular imaging system according to any one of claims 1-6 in the diagnosis of prostate cancer.

8. The application of the intracellular imaging system according to any one of claims 1-6 in defining the surgical boundaries of prostate cancer.

9. The application of the intracellular imaging system according to any one of claims 1-6 in the evaluation of the efficacy of prostate cancer treatment.

10. The use of the intracellular imaging system according to any one of claims 1-6 in the screening of candidate drugs for prostate cancer.

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