Fluorescent probe for detecting G-quadruplex

Abstract

The invention discloses a fluorescent probe for detecting G-quadruplex, and belongs to the technical field of biomedical detection. The fluorescent probe comprises a fusion protein, the fusion protein comprises a peptide fragment A, the peptide fragment A can be specifically combined with a G-quadruplex, and the amino acid sequence of the peptide fragment A is as shown in SEQ ID NO.1; and the cyclic rearrangement fluorescent protein is operably connected with the peptide fragment A. The fluorescent probe not only can specifically recognize in an in-vitro sample and correspond to various G4 conformations, but also can realize high-resolution imaging in living cells, promotes basic research of G4 functions, and opens up a new way for transforming medicine and clinical application.

Description

Technical Field

[0001] This application belongs to the field of biomedical detection technology, specifically relating to a fluorescent probe for detecting G-quadruplexes. Background Technology

[0002] G-quadruplexes (G4) are atypical nucleic acid secondary structures formed by guanine-rich DNA or RNA sequences linked by Hoogsteen hydrogen bonds. Their structural characteristic is the formation of a ring-like plane of four guanine molecules linked by Hoogsteen hydrogen bonds, with multiple planes stacked π-π to form a quadruplex structure. This non-classical nucleic acid structure exhibits significant conformational polymorphism (classified into parallel, mixed, and antiparallel topological types based on the orientation of the nucleic acid chains). Existing research has shown that G4 is specifically enriched in key functional regions such as telomeres, gene promoter regions, ribosomes and mitochondria, and introns and coding regions of mRNA. Its dynamic formation and unwinding in specific regions of the genome play a crucial role in key biological processes such as gene expression regulation, DNA replication, transcription, and telomere maintenance, making it a potential intervention target for various major diseases, including cancer, neurodegenerative diseases, and diabetes. Therefore, accurate detection and visualization of G4 are essential for understanding the occurrence and progression of G4-related diseases, providing important evidence for early diagnosis, treatment intervention, and drug development.

[0003] Currently, methods for detecting G-quadruplexes mainly include fluorescent probe-based detection techniques and antibody detection techniques. However, these methods suffer from insufficient specificity and sensitivity in complex biological matrices, making accurate detection of G4 difficult. Furthermore, they cannot achieve dynamic monitoring of G4 within living cells (such as rapid assembly under stress or pathological dissociation). In summary, existing methods face significant challenges in terms of dynamic monitoring of living cells, high-resolution imaging, and scalability.

[0004] Therefore, there is an urgent need to develop a non-invasive, highly specific detection tool that can achieve real-time detection and dynamic detection of G4 in living cells, thereby overcoming the static limitations of fixed samples, avoiding non-specific interference from chemical probes, and achieving subcellular spatial resolution and millisecond-level temporal sensitivity, so as to fully capture the complete spatiotemporal dynamic process of G4-DNA from formation to functional execution in living systems. Summary of the Invention

[0005] In view of this, the primary objective of this application is to provide a fluorescent probe for detecting G-quadruplexes. A fluorescent probe for detecting G-quadruplexes has been developed based on cyclic rearrangement fluorescent proteins. This fluorescent probe can specifically recognize and respond to multiple G4 conformations and can achieve high-resolution imaging in living cells, thereby promoting basic research on G4 function and opening up new avenues for translational medicine and clinical applications.

[0006] To achieve the above objectives, this application adopts the following technical solution: The first aspect of this application discloses a fluorescent probe for detecting G-quadruplexes, comprising a fusion protein, said fusion protein including: Peptide A, wherein peptide A is capable of specifically binding to G-quadruplexes, and the amino acid sequence of peptide A is shown in SEQ ID NO. 1; And a cyclic rearranged fluorescent protein, which is operatively linked to said peptide A.

[0007] This application develops a fluorescent probe for detecting G-quadruplexes based on circular rearranged fluorescent proteins. By reconnecting the N-terminus and C-terminus of the circular rearranged fluorescent protein to form a closed circular structure, and introducing the G4-DNA specific binding domain RHAU23 (DHX36 segment 53-70) near the fluorophore, a specific response to the G4-DNA structure is achieved by inducing conformational rearrangement.

[0008] In this application, peptide A is the G4-DNA specific binding domain RHAU23 (DHX36 53-70), whose amino acid sequence is shown in SEQ ID NO.1, and it can specifically bind to G-quadruplexes.

[0009] As described in this application, "circular rearranged fluorescent protein" refers to a genetically engineered fluorescent protein variant in which the N-terminus and C-terminus of the original fluorescent protein are "cut" at specific positions and interchanged to form a new circular structure. In this application, the circular rearranged fluorescent protein is not particularly limited and can be one well-known in the art. Specific examples include, but are not limited to, circular rearranged green fluorescent protein (cpGFP), circular rearranged yellow fluorescent protein (cpYFP), circular rearranged red fluorescent protein (cpRFP), circular rearranged blue fluorescent protein (cpBFP), and circular rearranged cyan fluorescent protein (cpAmCyan). As a preferred example, the circular rearranged fluorescent protein is circular rearranged yellow fluorescent protein (cpYFP), whose amino acid sequence is SEQ ID NO.2.

[0010] As described in this application, a "fusion protein" refers to a genetic engineering technique that links two or more gene sequences from different sources together and expresses them in a host cell to form a single polypeptide chain. In this application, it specifically refers to a fusion protein formed by peptide A and a cyclic rearranged fluorescent protein.

[0011] As described in this application, "operably linked" refers to the connection of multiple polypeptide (or nucleotide) elements in a functional relationship. In this application, when a polypeptide element is placed at a position that has a functional relationship with another polypeptide or protein, they are operably linked. It is understood that "operably linked" can be a continuous connection or a spaced connection, which can be designed as needed, and those skilled in the art have the ability to do so.

[0012] In some specific examples, the cyclic rearranged fluorescent protein is inserted into the amino acid sequence of peptide A, dividing peptide A into two parts, A1 and A2, thereby forming the structure shown in formula (I): Formula (I): A1—linker1—C—linker2—A2.

[0013] In this context, C represents a cyclic rearranged fluorescent protein, and linker1 and linker2 are either independent linking peptides or absent. In some preferred examples, both linker1 and linker2 are absent.

[0014] As described in this application, the term "connecting peptide" refers to a short amino acid sequence that covalently links two or more functional protein domains or subunits. Its core function is spatial separation, ensuring that the connected parts can fold independently and perform their respective functions without interfering with each other. The specific design or selection can be made as needed.

[0015] As a preferred example, the cyclic fluorescent protein is inserted between the m-th to m+1-th amino acids of peptide A, where m is any integer between 6 and 11; preferably, m is 6, 7, 10, or 11.

[0016] As a specific example, the amino acid sequence of the fusion protein is shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

[0017] In other specific examples, peptide A is operatively linked to the N-terminus and / or C-terminus of the cyclic rearranged fluorescent protein to form a structure as shown in formula (II): Formula (II): B1—linker3—C—linker4—B2; In this context, C represents a cyclic rearranged fluorescent protein, linker3 and linker4 are either independent linking peptides or absent; B1 is peptide A or absent, B2 is peptide A or absent, and at least one of B1 and B2 is present.

[0018] As a preferred example, linker3 and linker4 are flexible linker peptides that can be designed according to functional requirements. As a specific example, in formula (II), the amino acid sequences of linker3 and linker4 are GTGSGA (SEQ ID NO.7).

[0019] As a specific example, the amino acid sequence of the fusion protein is shown in SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10.

[0020] Furthermore, it is understood that the fluorescent probe also includes functional domains that are operatively linked to the fusion protein, preferably via adaptor peptides. Specific functional domains are not particularly limited and can be designed according to actual application requirements. Specific examples of the functional domains include, but are not limited to, at least one of the following: nuclear localization signal peptides, mitochondrial localization signal peptides, reporter proteins, detection tag proteins, and purification tag proteins; they can be one or more of these.

[0021] As described in this application, the term "nuclear localization signal peptide" refers to a short amino acid sequence that guides the active transport of proteins carrying that sequence from the cytoplasm to the nucleus through the nuclear pore complex. It is a key element ensuring that tool proteins enter the nucleus to perform their functions.

[0022] The term "mitochondrial localization signal peptide" refers to a short amino acid sequence that guides the active transport of proteins carrying this sequence from the cytoplasm into the mitochondria via the mitochondrial membrane transport complex. It is a key element ensuring that nuclear-coded proteins enter the mitochondria to perform their functions.

[0023] The term "reporter protein" refers to a protein whose expression or activity can be easily detected quantitatively or qualitatively; specific examples include fluorescent proteins and luciferases.

[0024] The term "detection tag protein" refers to a known peptide or protein segment that can be recognized by specific ligands (such as antibodies), fused to a target protein for the purpose of localization, tracing, or quantitative analysis of the target protein. Specific examples include, but are not limited to, c-Myc tags, HA tags, Flag tags, and GFP tags.

[0025] The term "purification-tagged protein" refers to a known peptide or protein segment that binds reversibly with high affinity to a specific chromatographic medium, fused to a target protein for the rapid and efficient separation and purification of the target protein from complex mixtures. Specific examples include, but are not limited to, histidine tags (His-tag), glutathione S-transferase tags (GST-tag), maltose-binding protein tags (MBP-tag), and streptavidin tags (Strep-tag).

[0026] The term "connector peptide" refers to a relatively simple amino acid sequence used to link two functional modules. Specific examples include, but are not limited to, one or more of LE, TR, SAG, GT, SA, EF, T, R, L, E, S, A, and G. These can be designed or selected as needed.

[0027] As a specific example, the amino acid sequence of the nuclear localization signal peptide is shown in SEQ ID NO.11; and / or, the amino acid sequence of the mitochondrial localization signal peptide is shown in SEQ ID NO.12; and / or, the detection tag protein is a 3-flag tag with an amino acid sequence as shown in SEQ ID NO.13 or an HA tag with an amino acid sequence as shown in SEQ ID NO.14; and / or, the purified tag protein is a 6×his tag.

[0028] As a specific example, the amino acid sequence of the fluorescent probe is any one of SEQ ID NO.15-23.

[0029] The second aspect of this application discloses the use of the fluorescent probe as described in the first aspect of this application in the preparation of a kit for detecting G-quadruplexes.

[0030] The third aspect of this application discloses a kit for detecting G-quadruplexes, containing the fluorescent probe described in the first aspect of this application. The kit may also include auxiliary reagents required for different detection technologies, such as buffer solutions and enzymes, which can be configured as needed by those skilled in the art and are therefore not particularly limited.

[0031] The beneficial effects of this application are: This application presents a fluorescent probe for detecting G-quadruplexes based on cyclic rearranged fluorescent proteins. This probe is constructed using a gene-encoding strategy, eliminating the need for complex chemical synthesis, allowing for gradual preparation, and reducing costs. This probe can not only detect G-quadruplexes in in vitro samples but also achieve highly sensitive and specific imaging of G-quadruplexes in living cells and even in vivo environments, providing a powerful tool for functional research and clinical translation of G-quadruplexes. Attached Figure Description

[0032] Figure 1 This application shows a schematic diagram of the structure of the fluorescent probe G4-Flame in a preferred embodiment. Figure 1 (A) and a schematic diagram of the detection principle ( Figure 1 (B)

[0033] Figure 2 The image shows the spectrum of plasmid PET-22B-NLS-G4-Flame from Example 2.

[0034] Figure 3 This is a demonstration of the excitation wavelength results of the G4-Flame fluorescent probe in Example 2.

[0035] Figure 4 This demonstrates the results of the fluorescent probe specificity study in Example 3; Figure 4 In the image, A and B represent the spectral scans of G4-Flame binding to G4-DNA under excitation light of 485 nm and 405 nm, respectively. Figure 4 C and D in the image represent the spectral scans of G4-Flame combined with ssMUT under excitation light of 485 nm and 405 nm, respectively. Figure 4 E and F in the image represent the spectral scans of G4-Flame binding to dsDNA under excitation light of 485 nm and 405 nm, respectively.

[0036] Figure 5 This is a presentation of the results of the fluorescence probe sensitivity study in Example 3; Figure 5 In the figure, A represents the fluorescence intensity ratio at I485 / 405 when G4-Flame binds to parallel-G4. Figure 5 In the figure, B represents the fluorescence intensity ratio at I485 / 405 when G4-Flame binds to hybrid-G4. Figure 5 In the figure, C represents the fluorescence intensity ratio at I485 / 405 when G4-Flame binds to anti-parallel-G4.

[0037] Figure 6 This demonstrates the results of the EMSA study on the specificity of the fluorescent probe in Example 3. Figure 6 In diagram A, the EMSA results of G4-Flame binding to G4-DNA are displayed. Figure 6 B in the image shows the EMSA results of the combination of G4-Flame and ssMUT. Figure 6 The image in C represents the EMSA results of G4-Flame binding to dsDNA.

[0038] Figure 7 This demonstrates the specific determination results of different G4-Flame structures in Example 3. Figure 7 A in the diagram represents different G4-Flame structures. Figure 7In the figure, B represents the fluorescence intensity ratio at I485 / 405 when G4-Flame with different structures binds to ssMUT and dsDNA. Figure 7 In the figure, C represents the fluorescence intensity ratio at I485 / 405 when G4-Flame with different structures binds to parallel-G4, hybrid-G4, and anti-parallel-G4.

[0039] Figure 8 This illustrates the application of the G4-Flame probe in clinical patient testing as described in Example 4. Figure 8 In Figure A, the statistical results of G4-DNA concentration detected in the serum of cancer patients and normal patients are presented. Figure 8 In Figure B, the ROC curve for G4-DNA detection by G4-Flame is shown.

[0040] Figure 9 The map of the recombinant plasmid PCDH-NLS-FLAME-NEO constructed in the in vivo experimental part of Example 5 ( Figure 9 (A) and PCDH-MITO-FLAME-NEO map ( Figure 9 (B)

[0041] Figure 10 For the NLS-G4-Flame nuclear colocalization analysis in Example 5 ( Figure 10 (A); MITO-G4-Flame mitochondrial colocalization analysis ( Figure 10 (B)

[0042] Figure 11 The results of the intracellular MITO-G4-Flame sensor detection in Example 5 are shown below. Figure 11 Center A shows the results of the microscopic imaging. Figure 11 B represents the statistical results of the fluorescence intensity ratio.

[0043] Figure 12 The results of the intracellular NLS-G4-Flame sensor detection in Example 5 are shown below. Figure 12 Center A shows the results of the microscopic imaging. Figure 12 B represents the statistical results of the fluorescence intensity ratio.

[0044] Each experimental group included a blank control group (Ctrl); ns indicates no significant difference. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001. Detailed Implementation

[0045] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0047] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0048] Example 1 In this embodiment, the circular rearranged yellow fluorescent protein (cpYFP) is used as an example. The N-terminus and C-terminus of cpYFP are reconnected to form a closed circular structure, and the G4-DNA specific binding domain—peptide A, namely RHAU23 (DHX36 53-70 segment) is introduced near the fluorophore to construct a fluorescent probe.

[0049] The amino acid sequence of peptide A is HPGHLKGREIGMWYAKKQGQKNK (SEQ ID NO.1).

[0050] The amino acid sequence of cpYFP is as follows: YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVN GHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN (SEQ ID NO.2).

[0051] In some examples, cpYFP is inserted into positions 6 / 7, 7 / 8, 10 / 11, or 11 / 12 of peptide A to obtain fusion proteins with amino acid sequences as shown in SEQ ID NO. 3-6.

[0052] In other examples, peptide A was linked to the N-terminus and / or C-terminus of cpYFP to obtain fusion proteins with amino acid sequences as shown in SEQ ID NO. 8-10, with the linker peptide being GTGSGA (SEQ ID NO. 7).

[0053] The above fusion protein was linked to the nuclear localization sequence PKKKRKV (SEQ ID NO.11), the mitochondrial localization sequence SVLTPLLLRGLTGSARRLPVPRAK (SEQ ID NO.12), the 3Flag tag DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO.13), the HA tag YPYDVPDYA (SEQ ID NO.14), or the His tag HHHHHH, as well as a suitable adaptor peptide, to obtain the fluorescent probes for detecting G-quadruplexes as shown in Table 1.

[0054] Table 1. Fluorescent probes for detecting G-tetrachain

[0055]

[0056] Taking G4-Flame as an example, its structure and mechanism of action are as follows: Figure 1 As shown, by reconnecting the N-terminus and C-terminus of the circularly rearranged fluorescent protein to form a closed circular structure, a G4-DNA specific binding domain is introduced near the fluorophore, inducing conformational rearrangement and thus achieving a specific response to the G4-DNA structure.

[0057] Example 2: Preparation and Excitation Light Determination of G4-Flame 2.1 Preparation of G4-Flame (1) Construct a recombinant plasmid containing the target protein G4-Flame Using pET-22b as a vector, a third-party institution was commissioned to insert the nucleotide sequence (SEQ ID NO. 24) encoding the target protein G4-Flame into the multiple cloning site (MCS) of the vector pET-22b between the Nhe I and BamHI restriction sites, obtaining the recombinant plasmid PET-22B-NLS-G4-Flame (plasmid map shown). Figure 2 ).

[0058] (2) Protein purification BL21(DE3) competent cells containing the recombinant plasmid PET-22B-NLS-G4-Flame expressing the target protein (G4-Flame) were seeded into LB medium (10 g tryptone, 5 g yeast extract, 10 g sodium chloride) containing 50 μg / ml streptomycin and cultured. The overnight culture was then used to inoculate 1 L of LB medium containing 50 μg / ml streptomycin. Protein expression was induced by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and the culture was then incubated overnight at 16°C with shaking at 200 rpm. Cell lysis was performed using Tiechui lysis buffer (ACE Biosciences, catalog number BR0005) at 4°C on a rotary mixer for 20 minutes. After lysis, the lysate was centrifuged at 4°C and 16000g for 10 minutes, and the supernatant was filtered through a 0.45 μm filter for clarification. The clarified lysis supernatant was loaded onto a Ni-NTA agarose affinity chromatography column pre-equilibrated with binding buffer. The column was first washed with five column volumes of binding buffer containing 10 mM imidazole (pH 8.0). The target protein was then eluted with binding buffer containing 200 mM imidazole (pH 8.0), and the eluent was replaced with G4 buffer (10 mM Tris-HCl, 140 mM KCl) via dialysis. Protein concentration was determined using Amicon Ultra-15 centrifuge tubes (Millipore, catalog number UFC9010) with a molecular weight cutoff of 30 kDa, and quantified using NanoDrop. Protein purity was assessed by SDS-PAGE electrophoresis. Samples were ultimately stored at -80°C for subsequent analysis.

[0059] 2.2 Selection of excitation and emission light of G4-Flame fluorescent probe The spectral changes of the G4-Flame probe before and after the addition of parallel G4-DNA (see Table 2 for the specific sequence) were determined using a UV spectrophotometer.

[0060] Table 2 DNA Nucleic Acid Sequences

[0061] Note: Double-stranded DNA is formed by annealing two strands. In addition, G4-DNA structure is formed by annealing the above three G4-DNA nucleic acid sequences.

[0062] The results are as follows Figure 3 As shown, after the addition of G4-DNA, the spectral peak of the G4-Flame probe at 405 nm increased, while the spectral peak at 485 nm decreased. Therefore, 405 nm and 485 nm were chosen as the excitation wavelengths for the G4-Flame probe.

[0063] Example 3: Specificity and Sensitivity of Fluorescent Probes 3.1 G4-Flame Specificity Study The probe, with a final concentration of 2 μM, was mixed with parallel G4-DNA, single-stranded DNA (ssMUT), and double-stranded DNA (dsDNA) with a final concentration of 10 μM (see Table 2 for specific sequences). The total mixture volume was 100 μL. The results were obtained using a black-background microplate (Sangon Biotech, catalog number F605034), and the fluorescence spectra at excitation light were measured at 485 nm and 405 nm.

[0064] The results are as follows Figure 4 As shown, when G4-Flame binds to G4-DNA, there is a significant difference in the emitted light at 520 nm at excitation light positions of 485 nm and 405 nm. Specifically, the light intensity at 405 nm increases with increasing G4-DNA concentration, while the light intensity at 485 nm decreases with increasing G4-DNA concentration, exhibiting a larger change in fluorescence intensity. However, the change in fluorescence intensity is smaller when binding to single-stranded and double-stranded DNA. This indicates that the G4-Flame disclosed in this application has a strong recognition ability for G4-DNA.

[0065] 3.2 G4-Flame Sensitivity Study Using excitation wavelengths of 485 nm and 405 nm, respectively, and emission wavelengths of 520 nm, the fluorescence intensity of G4-Flame probe with a final concentration of 500 nM was measured after mixing with 10 μM, 5 μM, 1 μM, 500 nM, 100 nM, 50 nM, and 500 pM of parallel, mixed, and antiparallel G4-DNA (specific sequences are shown in Table 2).

[0066] The results are as follows Figure 5 As shown, the I485 / 405 ratio continuously decreases with increasing G4-DNA concentration, indicating that the binding of the G4-Flame probe to G4-DNA can reach the nanomolar level. This means that G4-Flame has the ability to recognize all three G4-DNA structures, and this recognition ability can reach the nanomolar level.

[0067] 3.3 EMSA (Electrophoretic Mobility Variation Assay) Study on the Specificity of the Fluorescent Probe in Example 1 (1) Prepare EMSA adhesive according to the formula in Table 3: Table 3 Formulation of 9% Non-Modified Polyacrylamide Adhesive

[0068] (2) Binding: Take G4-Flame probes with final concentrations of 6 μM, 0.6 μM, and 0.06 μM respectively and incubate them with nucleic acid labeled with cy5.5 at a final concentration of 10 nM for 30 minutes at room temperature. The system is shown in Table 4.

[0069] Table 4

[0070] The binding buffer components are shown in Table 5: Table 5 Binding buffer components

[0071] (3) Electrophoresis: Prepare 1L of electrophoresis buffer (0.5×TBE, 20mM KCL), install the prepared gel into the vertical electrophoresis tank, and add electrophoresis buffer to cover the top edge of the gel plate; load the prepared samples, add an equal volume of 1×SDS to the blank wells to fill the gaps; electrophoresis at a constant voltage of 100V for 90 minutes and then stop. Keep the gel in an ice bath throughout the electrophoresis process.

[0072] (4) Development: Chemiluminescence developing instrument for developing and photographing.

[0073] The results are as follows Figure 6 As shown, the G4-Flame probe binds to G4-DNA, with the band blocked at the top of the gel. It does not bind to single-stranded or double-stranded DNA, with the band located at the bottom of the gel. This demonstrates the specificity of the G4-Flame probe.

[0074] 3.4 Sensitivity and Specificity Study of Different Fluorescent Probes (1) Recombinant plasmids containing the target protein were constructed according to the method in Example 2, and the protein was purified to obtain the fluorescent probes G4-Flame-1, G4-Flame-2, G4-Flame-3, G4-Flame-4, G4-Flame-N and G4-Flame-C in Table 1.

[0075] (2) Fluorescence intensity measurement: Take 2 μM G4-Flame-1, G4-Flame-2, G4-Flame-3, G4-Flame-4, G4-Flame-N, G4-Flame-C probes and mix them with 10 μM nucleic acid to prepare a 100 μL solution. Place it in a black-background microplate for detection. Record the fluorescence intensity at excitation wavelengths of 485 nm and 405 nm and emission wavelength of 520 nm and calculate it.

[0076] The results are as follows Figure 7 As shown, the G4-Flame-N and G4-Flame-C probes do not bind to either single-stranded or double-stranded DNA, but they have a strong recognition function for parallel G4-DNA and can be used for the detection of specific parallel G4-DNA.

[0077] Example 4 In this embodiment, the G4-Flame probe was applied to a tumor patient to verify its potential clinical application. The specific steps are as follows: (1) Take 5 μL of clinical patient serum (including serum from 42 tumor patients and 27 normal patients) and mix it with 5 μL of G4-Flame probe with a final concentration of 500 nM. (2) Place it in a 384-well microplate for fluorescence intensity detection and calculation.

[0078] The results are as follows Figure 8 As shown, analysis and calculations revealed that the G4-DNA content in the serum of tumor patients was higher than that in the serum of healthy individuals, consistent with current literature reports. Furthermore, the receiver operating characteristic (ROC) curve demonstrated the excellent detection efficacy of this fluorescent probe, indicating its significant potential for clinical applications.

[0079] The serum samples from the clinical patients in this embodiment were all collected from the First Affiliated Hospital of the University of Science and Technology of China (Anhui Provincial Hospital), among which the tumor patients were mainly liver cancer patients.

[0080] Example 5: Detection of intracellular G4 5.1 Construction of recombinant plasmids Using PCDH-NEO as a vector, a third-party institution was commissioned to insert the target genes NLS-3FLAG-FLAME (nucleotide sequence SEQ ID NO. 25) and MITO-HA-FLAME (nucleotide sequence SEQ ID NO. 26) between the Nhe I and BamHI restriction sites in the multiple cloning site (MCS) of the vector, respectively, to construct the recombinant plasmid PCDH-NLS-3FLAG-FLAME-NEO. Figure 9 (A) and PCDH-MITO-HA-FLAME-NEO ( Figure 9 (B)

[0081] 5.2 Plasmid transformation and extraction (1) Plasmid transformation: Place the ligation product on ice, take out competent Escherichia coli (DH5α) and thaw on ice, add 10 μL of ligation product to 20 μL of competent cells, pipette to mix evenly, incubate on ice for 20 min; heat shock at 42℃ for 90 s; incubate on ice for 2 min; add 100 μL of antibiotic-free LB medium, incubate at 37℃ for 30 min; spread the bacterial solution evenly on solid LB medium with the corresponding antibiotic using a spreader, and incubate at 37℃ for 12-16 h.

[0082] (2) Plasmid extraction: Select vigorous colonies and incubate them in liquid LB medium with appropriate antibiotics at 37°C for 12 h; take 500 μL of the bacterial culture, add an equal volume of sterilized 30% glycerol, mix well, and freeze at -80°C for preservation; take the above-cultured bacterial culture, centrifuge at 3000 rpm at room temperature for 8 min, and discard the supernatant. Perform plasmid extraction and determine the concentration according to the instructions of the AxyPrep endotoxin-free plasmid mini kit (uelandy, catalog number UE-MN-P), and store the obtained plasmid at 4°C.

[0083] 5.3 Plasmid transfection (1) Transfer the cells to be transfected (HEK293T) to the cell culture plate in advance and culture them to a cell density of 60%-80%. Replace the medium with fresh medium before transfection.

[0084] (2) The plasmid transfection was performed using the liposome transfection method. According to Table 6, an appropriate amount of plasmid to be transfected (the amount of plasmid is shown in the table below) and Opti-MEM medium were added to the EP tube, gently blown to mix, and incubated at 37℃ for 5 min.

[0085] Table 6. Quantities of reagents used for plasmid transfection

[0086] (3) Add an appropriate amount of Lipo2000 reagent (refer to Table 2 for dosage) and Opti-MEM medium to a sterile EP tube, mix well, and let stand in a 37℃ incubator for 5 min.

[0087] (4) Add the plasmid-Opti-MEM mixture to the Lipo2000-Opti-MEM mixture, mix by blowing and aspiration, and let stand at room temperature for 20 min.

[0088] (5) Add the above-prepared transfection mixture to a cell culture dish and incubate at 37°C in a CO2 incubator.

[0089] (6) Replace with fresh culture medium 8-12 hours after transfection and continue culturing.

[0090] (7) 36-48 hours after transfection, collect the cell supernatant as the target cell line for virus infection.

[0091] (8) Transfer the target cell lines (PLC, SNU449, HCCLM3) to cell culture plates in advance and incubate them to a cell density of 40%-60% before infection (virus: normal culture medium = 1:1). (9) Twelve hours after infection, the cells were screened using a medium containing G418 (50 μg / mL, Beyotime, catalog number ST081). After 5-7 days of screening, the target cell lines that stably expressed PCDH-NLS-3FLAG-FLAME-NEO and PCDH-MITO-HA-FLAME-NEO were obtained.

[0092] 5.4 Cellular Immunofluorescence (1) Cells expressing nuclear localization signal marker G4-Flame sensor (NLS-G4-Flame) or mitochondrial localization marker G4-Flame sensor (Mito-G4-Flame) were seeded in confocal culture dishes.

[0093] (2) After culturing for 24 hours, the cells were washed twice with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde for 20 minutes at room temperature.

[0094] (3) Permeabilize the cells with 0.2% Triton X-100 and block them with 5% bovine serum albumin (BSA).

[0095] (4) Stain the cells according to the specific experimental purpose. (5) Perform fluorescence imaging. For cells without special staining requirements, G4-Flame cells were directly subjected to fluorescence imaging after fixation. Finally, images were acquired using a Leica STED confocal microscope.

[0096] The results are as follows Figure 10 As shown, cells expressing G4-Flame sensors with nuclear localization signal markers can colocalize with PI (nuclear localization markers), and cells expressing G4-Flame sensors with mitochondrial localization signal markers can colocalize with ATP5A1 (mitochondrial localization protein), thus demonstrating the successful construction of cells with subcellular localization G4-Flame sensors.

[0097] 5.5 Application of the intracellular MITO-G4-Flame sensor (1) Cells (SUN449) expressing mitochondrial localization G4-Flame sensor were seeded in confocal dishes and G4-DNA stabilizer PDS 100μM was added to the cells for two hours, four hours, eight hours and ten hours respectively.

[0098] (2) After removing the culture medium, fix with 4% paraformaldehyde at room temperature for 20 minutes.

[0099] (3) Images were acquired using a Leica STED confocal microscope.

[0100] The results are as follows Figure 11As shown, cells expressing the G4-Flame sensor, a marker of mitochondrial localization signaling, showed a significant decrease in I485 / 405 after the addition of PDS, consistent with the expected results.

[0101] 5.6 Application of Intracellular NLS-G4-Flame Sensor (1) Cells expressing the G4-Flame sensor (HEK 293T) with nuclear localization signal were seeded in confocal dishes and cell cycle regulation was achieved by adding thymidine (MCE, catalog number HY-N1150).

[0102] (2) S phase and G1 phase cells were fixed.

[0103] (3) Images were acquired using a Leica STED confocal microscope.

[0104] The results are as follows Figure 12 As shown, cells expressing the G4-Flame sensor, a nuclear localization signal marker, exhibited lower G4-DNA levels in S-phase cells and higher G4-DNA levels in G1-phase cells.

[0105] The above embodiments demonstrate that the G-quadruplex fluorescent probe disclosed in this application can detect G-quadruplexes with significant specificity and sensitivity. More importantly, it can not only detect G-quadruplexes in in vitro samples, but also achieve high-sensitivity and high-specificity imaging of G-quadruplexes in living cells and even in vivo environments.

[0106] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A fluorescent probe for detecting G-quadruplexes, characterized in that, Includes a fusion protein, said fusion protein comprising: Peptide A, wherein peptide A is capable of specifically binding to G-quadruplexes, and the amino acid sequence of peptide A is shown in SEQ ID NO.1; And a cyclic rearranged fluorescent protein, which is operatively linked to said peptide A.

2. The fluorescent probe as described in claim 1, characterized in that, The cyclic rearranged fluorescent protein is one of the following: cyclic rearranged yellow fluorescent protein, cyclic rearranged green fluorescent protein, cyclic rearranged red fluorescent protein, and cyclic rearranged blue fluorescent protein. Preferably, the cyclic rearranged fluorescent protein is a cyclic rearranged yellow fluorescent protein.

3. The fluorescent probe as described in claim 1 or 2, characterized in that, The cyclic rearranged fluorescent protein is inserted into the amino acid sequence of peptide A, dividing peptide A into two parts, A1 and A2, thereby forming the structure shown in formula (I): Formula (I): A1—linker1—C—linker2—A2; Alternatively, peptide A can be operatively linked to the N-terminus and / or C-terminus of the cyclic rearranged fluorescent protein, thereby forming a structure as shown in formula (II): Formula (II): B1—linker3—C—linker4—B2; In formulas (I) and (II), C represents a cyclic rearranged fluorescent protein, linker1, linker2, linker3 and linker4 are independently linking peptides or absent; B1 is peptide A or absent, B2 is peptide A or absent, and at least one of B1 and B2 is present.

4. The fluorescent probe as described in claim 3, characterized in that, In formula (I), the cyclic rearranged fluorescent protein is inserted between the m-th to m+1-th amino acids of peptide A, where m is any integer between 6 and 11; Preferably, m is 6, 7, 10 or 11.

5. The fluorescent probe as described in claim 4, characterized in that, In equation (I), both linker1 and linker2 are absent; Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.

6.

6. The fluorescent probe as described in claim 3, characterized in that, In formula (II), the amino acid sequences of linker3 and linker4 are shown in SEQ ID NO.7; Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.

10.

7. The fluorescent probe as described in claim 1, characterized in that, The fluorescent probe further includes a functional domain that is operatively linked to the fusion protein; The functional domains include at least one of the following: nuclear localization signal peptide, mitochondrial localization signal peptide, reporter protein, detection tag protein, and purification tag protein; Preferably, the amino acid sequence of the nuclear localization signal peptide is as shown in SEQ ID NO.11; and / or, the amino acid sequence of the mitochondrial localization signal peptide is as shown in SEQ ID NO.12; and / or, the detection tag protein is a 3-flag tag with an amino acid sequence as shown in SEQ ID NO.13 or an HA tag with an amino acid sequence as shown in SEQ ID NO.14; and / or, the purified tag protein is a 6×his tag; Preferably, the functional domain is linked to the fusion protein via a linker peptide; Preferably, the amino acid sequence of the linker peptide is selected from one or more of LE, TR, SAG, GT, SA, EF, T, R, L, E, S, A, and G.

8. The fluorescent probe as described in claim 7, characterized in that, The amino acid sequence of the fluorescent probe is any one of SEQ ID NO. 15-23.

9. Use of the fluorescent probe according to any one of claims 1-8 in the preparation of a kit for detecting G-quadruplexes.

10. A kit for detecting G-quadruplex, characterized in that, It contains the fluorescent probe according to any one of claims 1-8.

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