Fluorescent probe for G-quadruplex DNA detection

Abstract

The invention belongs to the technical field of G-quadruplex detection, and particularly relates to a fluorescent probe for G-quadruplex DNA detection, the fluorescent probe comprises a fluorescent probe compound, the fluorescent probe compound generates a fluorescent signal after being combined with a G-quadruplex, and qualitative and quantitative analysis can be performed on the G-quadruplex in cells; the fluorescent probe is (E)-1, 3, 3-trimethyl-2-(4-(piperidine-1-yl) styryl)-3H-indole-1-iodine, and the structural formula of the fluorescent probe is shown in the description. The probe provided by the invention has the advantages of simple preparation and easily available raw materials, and is stable in structure and convenient to store; the probe has relatively low biotoxicity, phototoxicity and photobleaching property; in practical application, the negative influence on organisms is small, and the safety and reliability of experiments can be ensured. Moreover, the probe has excellent light stability, can maintain stable performance under different illumination conditions, and provides powerful guarantee for long-term experimental observation and research.

Description

Technical Field

[0001] The invention relates to the technical field of G-quadruplex detection, in particular to a fluorescent probe for G-quadruplex DNA detection. Background Art

[0002] G-quadruplex is a high-order structure formed by the folding of DNA or RNA rich in tandem repeats of guanine (G). G-tetrad is the structural unit of G-quadruplex. Four Gs are connected by Hoogsteen hydrogen bonds to form a ring-shaped plane. Two or more layers of tetrads form quadruplexes through π-π stacking. The formation of G4 structure plays a vital regulatory role in a series of physiological processes in the body. A large number of studies have confirmed that the G4 structure in some promoter regions significantly affects the transcription and translation levels of genes. The G4 structure is widely believed to play a key function as a molecular switch. The process of its formation and disassembly involves a series of physiological processes such as signal transduction, cell apoptosis and cell proliferation. Therefore, specifically detecting the existence of G4 structure or monitoring its dynamic process is of great significance for in-depth research on the relevant biological functions of G4 structure and the development of anticancer drugs targeting G4 structure.

[0003] However, the current research on G4 only involves a few gene sequences, and there are still a large number of potential G4 sequences waiting to be confirmed through experiments. In addition, the dynamic changes of G4 in cells also need to be further explored. In view of this, it is particularly important to develop methods and tools that can quickly confirm the structure of G4. Small molecule probes for G4 detection have also become a research hotspot at this stage.

[0004] Fluorescent probes have developed rapidly in both theory and application, and are now widely used in many fields such as medicine, life sciences, and environmental sciences. However, these probes have problems such as low selectivity, poor biocompatibility, and background interference, which severely limit their in vivo and in vitro applications. Therefore, it is of great significance to design and synthesize small molecule fluorescent probes that are selective, sensitive, and biocompatible and can detect G4 in cells and in vivo. To this end, we hereby disclose a new type of G4 fluorescent probe and its detection method.

[0005] It should be noted that the above contents belong to the technical knowledge of the inventor and do not necessarily constitute prior art. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a fluorescent probe for G-quadruplex DNA detection, which solves the existing problems.

[0007] To achieve the above object, the present invention provides the following technical solution: a fluorescent probe for detecting G-quadruplex DNA, comprising:

[0008] A fluorescent probe compound, which generates a fluorescent signal after binding to a G-quadruplex, and can perform qualitative and quantitative analysis of the G-quadruplex in cells;

[0009] The fluorescent probe is (E)-1,3,3-trimethyl-2-(4-(piperidin-1-yl)phenylvinyl)-3H-indole-1-iodine, and its structure is shown below:

[0010]

[0011] In some of these embodiments, a G-quadruplex is a higher-order structure formed by folding DNA or RNA rich in tandem repeats of guanine (G);

[0012] G-tetrad is the structural unit of G-quadruplex, with four Gs connected by Hoogsteen hydrogen bonds to form a ring-shaped plane, and two or more layers of tetrads form a quadruplex through π-π stacking;

[0013] G-quadruplexes are involved in telomere end protection, DNA replication, transcription and translation, and can effectively regulate cell proliferation, apoptosis and aging, and the occurrence and development of tumors.

[0014] In some of the embodiments, the fluorescent probe compound has an electronic conjugation system and a rigid plane, and in the interaction with the G4-quadruplex structure, can be stacked on the tetrad plane of the G-quadruplex and has an interaction force with the G-quadruplex.

[0015] In some of the embodiments, the qualitative and quantitative analysis of G-quadruplexes is that the G-quadruplexes can generate fluorescence of a specific wavelength after binding to the probe compound, the fluorescence intensity is linearly related to the G-quadruplex process, and no fluorescence is generated when the substrate is non-G-quadruplex.

[0016] In some embodiments, the binding experiment process in which the probe compound generates a fluorescent signal after binding to a G-quadruplex is performed in the following specific steps:

[0017] Step 1: dissolving the G-quadruplex to be tested in a buffer solution with a pH value of 7.2-7.4 to obtain solution A; dissolving the trimethyl indole compound in dimethyl sulfoxide, and then diluting it with a buffer solution with a pH value of 7.2-7.4 to obtain solution B;

[0018] Step 2: Mix solution A and solution B in step 1 to obtain a mixed solution, in which the molar ratio of G-quadruplex to trimethylindole compound is 10:1, and then perform fluorescence spectrum analysis on the mixed solution and observe it under ultraviolet light.

[0019] In some embodiments, the buffer is a Tris-hydrochloric acid buffer, and the pH value is regulated by hydrochloric acid.

[0020] In some embodiments, when the mixed solution is subjected to fluorescence spectral analysis, if the fluorescence emission intensity enhancement factor of the mixed solution at 575nm to 625nm is greater than 1 times compared with solution B, it can be determined that the nucleic acid to be tested is a G-quadruplex.

[0021] In some of the embodiments, when the mixed solution is subjected to fluorescence spectroscopy for quantification, the fluorescence emission intensity has a limit of detection (LOD) of less than 40 nM for G-quadruplex, and the G-quadruplex of the analyte can be detected in the background signal.

[0022] Compared with the prior art, the present invention provides a fluorescent probe for G-quadruplex DNA detection, which has the following beneficial effects:

[0023] 1. A fluorescent probe for G-quadruplex DNA detection. The probe provided by the present invention has the advantages of simple preparation and readily available raw materials, and at the same time, its structure is stable and easy to store;

[0024] 2. This fluorescent probe for G-quadruplex DNA detection has low biotoxicity, phototoxicity and photobleaching. In practical applications, it has little adverse effect on organisms, which can ensure the safety and reliability of the experiment. In addition, the probe has excellent photostability and can maintain stable performance under different lighting conditions, providing a strong guarantee for long-term experimental observation and research;

[0025] At the same time, the probe has good water solubility and can be fully dissolved in water, which is convenient for application in various aqueous environments. It also has good cell membrane permeability and can easily pass through the cell membrane and enter the cell for detection and research, which provides convenience for research in the field of cell biology.

[0026] Its spectral range is sufficiently different from that of biological samples. This feature enables the probe to have a lower fluorescence background in solutions and cells without G4, effectively avoiding interference and improving the accuracy and specificity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the hydrogen spectrum of the intermediate compound 2 in Example 1 of the present invention;

[0028] Figure 2 This is a schematic diagram of the hydrogen spectrum of probe compound 4 in Example 2 of the present invention;

[0029] Figure 3 It is a schematic diagram of the fluorescence intensity values ​​when the probe compound 4 of the present invention is incubated with DNA sequences with different secondary structures including single-stranded DNA, double-stranded DNA and G4-stranded DNA;

[0030] Figure 4 Schematic diagram of G4 selectivity when the probe compound 4 of the present invention is incubated with DNA sequences with different secondary structures including single-stranded DNA, double-stranded DNA and G4 strand DNA;

[0031] Figure 5 The fluorescence spectrum of the probe compound 4 solution in the present invention when G-quadruplex Pu27 is added dropwise;

[0032] Figure 6 It is a trend diagram between the fluorescence intensity of the probe compound 4 at 525 nm and the concentration of the sample to be tested in the present invention;

[0033] Figure 7 Schematic diagram of the linear synthesis equation of different G-quadruplex DNA titration probe compounds 4 of the present invention;

[0034] Figure 8 Schematic diagram of the fluorescence response value of the probe compound 4 after gradually adding double-stranded ct-DNA to a mixed solution of 1 μM probe compound 4 and 1 equivalent of G-quadruplex Pu27 (A) or Bcl-2 (B) of the present invention, and the fluorescence curve of the probe compound 4 solution dripping with G-quadruplex Pu27 (C) or Bcl-2 (D) in the presence or absence of ct-DNA;

[0035] Fig. 9 Schematic diagram of the fluorescence imaging of the probe compound 4 (1.0 μM) after co-incubation with fixed Siha cells and the fluorescence intensity after adding PDS (0, 1.0, 2.0 μM) in the present invention, the fluorescence imaging of the probe compound 4 (2.0 μM) co-incubated with Siha cells and the fluorescence imaging experimental state after DNase and RNase digestion. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] It should be understood that the step numbers used in this article are only for the convenience of description and are not intended to limit the order in which the steps are executed.

[0038] It should be understood that the terms used in the present specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the present specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are intended to include plural forms.

[0039] The terms “include” and “comprising” indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0040] The term "and / or" means and includes any and all possible combinations of one or more of the associated listed items.

[0041] See also Figure 1-9 , a fluorescent probe for G-quadruplex DNA detection, comprising:

[0042] A fluorescent probe compound, which generates a fluorescent signal after binding to a G-quadruplex and can perform qualitative and quantitative analysis of the G-quadruplex in cells;

[0043] The fluorescent probe is (E)-1,3,3-trimethyl-2-(4-(piperidin-1-yl)phenylvinyl)-3H-indole-1-iodine, and its structure is shown below:

[0044]

[0045] It should be noted that the G-quadruplex is a higher-order structure formed by folding DNA or RNA rich in tandem repeats of guanine (G);

[0046] The G-tetrad is a structural unit of a G-quadruplex, where four Gs are connected by Hoogsteen hydrogen bonds to form a ring-shaped plane, and two or more layers of tetrads form a quadruplex through π-π stacking;

[0047] The G-quadruplex is involved in telomere end protection, DNA replication, transcription and translation, and can achieve effective regulation of cell proliferation, apoptosis and aging, and the occurrence and development of tumors;

[0048] The fluorescent probe compound has an electronic conjugation system and a rigid plane, and can be stacked on the G-quadruplex tetrad plane in the interaction with the G4-quadruplex structure, and has an interaction force with the G-quadruplex;

[0049] The qualitative and quantitative analysis of the G-quadruplex is that the G-quadruplex can generate fluorescence of a specific wavelength after binding to the probe compound, the fluorescence intensity is linearly related to the G-quadruplex process, and no fluorescence is generated when the substrate is a non-G-quadruplex;

[0050] The specific steps of the binding experiment process in which the probe compound generates a fluorescent signal after binding to a G-quadruplex are as follows:

[0051] Step 1: dissolving the G-quadruplex to be tested in a buffer solution with a pH value of 7.2-7.4 to obtain solution A; dissolving the trimethyl indole compound in dimethyl sulfoxide, and then diluting with a buffer solution with a pH value of 7.2-7.4 to obtain solution B;

[0052] Step 2: Mixing solution A and solution B in step 1 to obtain a mixed solution, wherein the molar ratio of the G-quadruplex to the trimethylindole compound in the mixed solution is 10:1, and then performing fluorescence spectrum analysis on the mixed solution and observing it under ultraviolet light;

[0053] The buffer is a Tris-hydrochloric acid buffer, and the pH value is regulated by hydrochloric acid;

[0054] When the mixed solution is subjected to fluorescence spectral analysis by fluorescence spectral analysis, if the fluorescence emission intensity enhancement factor of the mixed solution at 575nm to 625nm is greater than 1 times compared with solution B, it can be determined that the nucleic acid to be tested is a G-quadruplex;

[0055] The fluorescence emission intensity is when the mixed solution is subjected to fluorescence spectrum analysis for quantification, and its limit of detection (LOD) for G-quadruplex is lower than 40 nM, and the G-quadruplex of the test object can be detected in the background signal.

[0056] Example 1: Synthesis of Compound 2, the structure of which is shown below:

[0057]

[0058] Under nitrogen, 2,3,3-trimethyl-3H-indole (0.5 g, 3.14 mmol) and iodomethane (1.4827 g, 12.56 mmol) were dissolved in acetonitrile and refluxed at 85°C. After cooling to room temperature, a large amount of solid precipitate was obtained. The solid was filtered through a Buchner funnel, washed three times with a mixed solvent of petroleum ether and ethyl acetate (v / v=3:1), and then dried in vacuo. The product was compound 2 with a yield of 86%. 1H NMR (400 MHz, DMSOd6) δ7.94-7.80 (m, 2H), 7.65-7.57 (m, 2H), 3.96 (d, J=1.1 Hz, 3H), 2.76 (d, J=1.0 Hz, 3H), 1.52 (s, 6H), which is as follows:

[0059]

[0060] Example 2: Synthesis of probe compound 4:

[0061] Compound 2 (130 mg, 0.454 mmol) and 4-piperidin-1-yl-benzaldehyde (103 mg, 0.5448 mmol) were placed in a flask, ethanol was added, and the mixture was heated to 85°C for 10 h under nitrogen. After the reaction, it was cooled to room temperature to obtain a large amount of solid precipitate. The solid was filtered through a Buchner funnel, washed three times with a mixed solvent of petroleum ether and ethyl acetate (v / v=3:1), and then vacuum dried to obtain compound 4 with a yield of 52.84%. 1H NMR (400MHz, DMSOd6) δ9.27(d,J=15.7Hz,1H),9.04(d,J=8.8Hz,2H),8.76(dd,J=7.4,1.3Hz,1H),8.69(d,J=7.9Hz,1H),8.54(td,J=7.7,1.3Hz, 1H),8.47(td,J=7.5,1.0Hz,1H),8.25(d,J=15.7Hz,1H),8.07(d,J=8.9 Hz, 2H), 4.95 (d, J = 4.4Hz, 3H), 4.57 (t, J = 5.3Hz, 4H), 2.69-2.51 (m, 6H).

[0062] Example 3: Detection of DNA samples:

[0063] 1. Sample Preparation

[0064] DNA samples: Nucleic acid samples were purchased from Sangon Biotech Co., Ltd. An appropriate amount of nucleic acid was dissolved in Tris-HC1 buffer (pH 7.2, 100 mM KC1), and the concentration was determined by ultra-micro-UV spectrometer. After heating at 95°C for 5 min, the solution was slowly cooled and annealed to room temperature as storage solution and stored at 4°C.

[0065] The DNA sample sequences tested include:

[0066]

[0067] Example 4: Selective fluorescence spectra of the synthetic compounds of Example 2 as fluorescent probes for different DNAs:

[0068] The synthesized compound 4 of Example 2 was diluted to a concentration of 100 μmol / L, and DNA of different sequences was added to measure the fluorescence intensity using a fluorescence spectrophotometer (slit width = 10, scanning speed = 200 nm, compound 4: Ex = 545 nm). Figure 3The fluorescence curves of the interaction between the compound synthesized in Example 2 and different types of DNA are shown in Table 1, which are Bcl-2, Pu27, Pu22, VEGF, ds26, ss26, mt1015, mt10252, mt12086, mt16250 and mt377, a total of 11 types. Figure 3 It can be seen that compound 4 has higher selectivity for G4 DNA than G4 mitochondrial DNA in in vitro experiments and has good nucleic acid discrimination.

[0069] Example 5. Fluorescence titration spectra of the synthetic compounds of Example 2 as fluorescent probes for different DNAs:

[0070] In the presence of compound 4 alone, there is a weak fluorescence emission at 610nm. After adding Pu27, the emission peak intensity at 610nm increased significantly, and with the increase in Pu27 concentration, the fluorescence continued to increase, reaching saturation when G4 reached 10 times the equivalent. Under ultraviolet light, after adding Pu27, obvious bright fluorescence was visible to the naked eye. Similarly, fluorescence titration experiments were also performed on other G4s, including ds26, Pu22, Bcl-2, and ss26. Figure 5 As shown in the figure, with the addition of different types of G4-chain DNA, the fluorescence intensity of compound 4 is significantly enhanced, and the fluorescence response of compound 4 to different G-quadruplexes has certain differences. Compound 4 has a high fluorescence response to parallel G4. Figure 5 It was found that the fluorescence intensity of Pu22 and Bcl-2 gradually increased, but not as much as that of Pu27. The fluorescence intensity of ds26 and ss26 did not increase significantly. This result shows that compound 4 can be used as a specific G-quadruplex fluorescent probe.

[0071] Example 6. Determination of the detection limit of the compound synthesized in Example 2 as a fluorescent probe:

[0072] Compound 4 was used to evaluate the detection performance of G4;

[0073] First, the detection limit of G4 in solution was studied. The limit of detection (LOD) is defined as the lowest concentration of the analyte that can be detected in the background signal when the signal reaches three times the noise. It is calculated according to the following equation: LOD = K × S / m. According to the recommendation of the International Union of Pure and Applied Chemistry (IUPAC), K is usually taken as 3; Sb represents the standard deviation of the noise signal of the solution of 20 times a specific concentration of compound 4; m is the slope of the standard curve, which can be obtained by fitting the straight line part of the fluorescence titration curve of compound 4 and G4. Parallel Bcl-2 and Pu27 were selected as the research objects. The detection limit of compound 4 for Pu27 is less than 30nM, and the detection limit for Bcl-2 is less than 40nM.

[0074] Example 8. Competitive fluorescence titration experiment of the compound synthesized in Example 2 as a fluorescent probe:

[0075] To verify the selectivity for G4, competitive fluorescence titration experiments were performed;

[0076] 1 μM compound 4 and 2 times equivalent Pu27 were placed in a mixed solution, and double-stranded ct-DNA was added. As the concentration of ct-DNA increased, the fluorescence intensity of the compound 4 and Pu27 complex was not disturbed. The same experiment was performed on Bcl-2, and the results were similar. When 200 μM ct-DNA was present, Pu27 was added to the compound 4 solution, and the fluorescence intensity increased steadily, similar to that in the absence of ct-DNA. In summary, compound 4 can specifically recognize G4 in a competitive environment.

[0077] Example 9. Cell imaging of the compound synthesized in Example 2:

[0078] Siha cells were cultured in high glucose DMEM (from Gibco) containing 10% FBS and penicillin / streptomycin in an incubator at 37°C with a 5% CO2 atmosphere. Cells were seeded into confocal glass bottom culture dishes (35.0 mm culture dishes, 20.0 mm bottom wells) and 2.0 mL of culture medium was added in the incubator until the cell density was approximately 50%, then washed three times with PBS and fixed with ice-cold ethanol. The cell culture dishes were stored in a -20°C refrigerator for 10 minutes, washed three times with PBS, and different concentrations of PDS ( Figure 7 A) or digestion with DNase I and RNase A ( Figure 7 B), after half an hour of incubation, the fluorescence images were recorded using a laser confocal fluorescence scanning microscope at a two-photon excitation wavelength of λex = 544.5 nm, and the collection wavelength was 565 ± 20 nm.

[0079] Each embodiment in this specification is described in a progressive manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment.

[0080] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fluorescent probe for detecting G-quadruplex DNA, characterized in that: include: A fluorescent probe compound, which generates a fluorescent signal after binding to a G-quadruplex and can perform qualitative and quantitative analysis of the G-quadruplex in cells; The fluorescent probe is (E)-1,3,3-trimethyl-2-(4-(piperidin-1-yl)phenylvinyl)-3H-indole-1-iodine, and its structure is shown below:

2. A fluorescent probe for G-quadruplex DNA detection according to claim 1, characterized in that: The G-quadruplex is a higher-order structure formed by folding DNA or RNA rich in tandem repeats of guanine (G); The G-tetrad is a structural unit of a G-quadruplex, where four Gs are connected by Hoogsteen hydrogen bonds to form a ring-shaped plane, and two or more layers of tetrads form a quadruplex through π-π stacking; The G-quadruplex is involved in telomere end protection, DNA replication, transcription and translation, and can achieve effective regulation of cell proliferation, apoptosis and senescence, and the occurrence and development of tumors.

3. A fluorescent probe for G-quadruplex DNA detection according to claim 1, characterized in that: The fluorescent probe compound has an electronic conjugation system and a rigid plane, and can be deposited on the G-quadruplex tetrad plane when interacting with the G4-quadruplex structure, and has an interaction force with the G-quadruplex.

4. A fluorescent probe for G-quadruplex DNA detection according to claim 1, characterized in that: The qualitative and quantitative analysis of the G-quadruplex is that the G-quadruplex can generate fluorescence of a specific wavelength after binding to the probe compound, the fluorescence intensity is linearly related to the G-quadruplex process, and no fluorescence is generated when the substrate is non-G-quadruplex.

5. A fluorescent probe for G-quadruplex DNA detection according to claim 1, characterized in that: The specific steps of the binding experiment process in which the probe compound generates a fluorescent signal after binding to a G-quadruplex are as follows: Step 1: dissolving the G-quadruplex to be tested in a buffer solution with a pH value of 7.2-7.4 to obtain solution A; dissolving the trimethyl indole compound in dimethyl sulfoxide, and then diluting with a buffer solution with a pH value of 7.2-7.4 to obtain solution B; Step 2: Mix solution A and solution B in step 1 to obtain a mixed solution, in which the molar ratio of G-quadruplex to trimethylindole compound is 10:1, and then perform fluorescence spectrum analysis on the mixed solution and observe it under ultraviolet light.

6. A fluorescent probe for G-quadruplex DNA detection according to claim 5, characterized in that: The buffer is a Tris-hydrochloric acid buffer, and the pH value is regulated by hydrochloric acid.

7. A fluorescent probe for G-quadruplex DNA detection according to claim 5, characterized in that: When the mixed solution is subjected to fluorescence spectral analysis by fluorescence spectral analysis, if the fluorescence emission intensity enhancement factor of the mixed solution at 575nm to 625nm is greater than 1 times compared with solution B, it can be determined that the nucleic acid to be tested is a G-quadruplex.

8. A fluorescent probe for G-quadruplex DNA detection according to claim 5, characterized in that: The fluorescence emission intensity is when the mixed solution is subjected to fluorescence spectrum analysis for quantification, and its limit of detection (LOD) for G-quadruplex is lower than 40 nM, and the G-quadruplex of the test object can be detected in the background signal.

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