Preparation method and application of biological stable compound

Abstract

The invention belongs to the field of fluorescent dyes, and particularly relates to a preparation method and application of a biological stable compound. The polymer has high stability in extracellular environment and intracellular environment under physiological pH and physiological temperature, and can be used in the fields of biochemical detection and living cell imaging. The invention also discloses a high-specificity and high-responsiveness nucleic acid substance inside and outside a cell and application of the probe in high-sensitivity detection of a living cell mitochondrial RNA-DNA (Ribonucleic Acid-Deoxyribose Nucleic Acid) hybrid. The fluorescent probe provided by the invention has the characteristics of strong color development, good light stability, good biological stability and the like. Meanwhile, the fluorescent probe is simple in preparation process, low in cost, stable in structure and convenient to store, and has a wide application space in biological function research of lysosome and mitochondria.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent dyes, and in particular relates to a preparation method of a biologically stable compound and application thereof. Background Art

[0002] Dyes are important chemical tool molecules, which are widely used in biological detection and other fields. Aryl methane compounds are a class of organic small molecule dyes with a long history, mainly including triarylmethane dyes and diarylmethane dyes, which are widely used in biology, environment and commodity dyeing.

[0003] However, for quite some time, the development of arylmethane dyes entered a period of relative stagnation. In recent years, with the development of science and technology, the application of arylmethane dyes in the field of life has regained attention. The latest research shows that arylmethane dyes have important applications in fluorescence imaging and biomacromolecule detection, suggesting that this type of molecule has great development potential in the biomedical field. Different from the widespread use of triarylmethane dyes in cells, although literature reports that diarylmethane dyes also have the potential to detect biomacromolecules, studies have shown that this type of dye molecule is very susceptible to attack by nucleophilic reagents, resulting in dye deterioration and difficulty in being widely used in the field of biological detection. So far, there are still no reports of diarylmethane dyes used in biological applications such as living cell imaging or in vivo imaging. Therefore, it is of great significance to further study and clarify the properties of diarylmethane dyes, solve the problem of their chemical stability, obtain new fluorescent dyes that can be widely used in the biological field, and dig out molecules with innovative biological applications. Summary of the invention

[0004] The first aspect of the present invention aims to provide a compound.

[0005] The object of the second aspect of the present invention is to provide a method for preparing the compound of the first aspect of the present invention.

[0006] The third aspect of the present invention aims to provide the use of the compound of the first aspect of the present invention in the preparation of a nucleic acid molecule detection product.

[0007] The fourth aspect of the present invention aims to provide a fluorescent probe.

[0008] The fifth aspect of the present invention aims to provide a biosensor.

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

[0010] The seventh aspect of the present invention aims to provide an application.

[0011] The eighth aspect of the present invention aims to provide a method for detecting nucleic acid.

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

[0013] The first aspect of the present invention provides a compound,

[0014] The structural formula of the compound is shown in formula (I):

[0015]

[0016] Wherein, R1 and R2 are each independently selected from an amino group or an amine substituent;

[0017] A - is an anion selected from I - , ClO4 - , TfO - 、SbF6 - PF6 - , Ts - or FSO2O - At least one of .

[0018] Preferably, the amino or amine substituent is selected from at least one of dimethylamino, diethylamino, pyrrolidin-1-yl, piperidin-1-yl, diphenylamino, and azetidin-1-yl.

[0019] In some embodiments of the present invention, R1 and R2 are dimethylamino.

[0020] In some embodiments of the present invention, A - trifluoromethanesulfonate (TfO - ), whose chemical name is bis(5-(dimethylamino)thiophen-2-yl)methylium triflate.

[0021] In some embodiments of the present invention, the compound is referred to as TiCa; its chemical structure is shown in Figure (II):

[0022]

[0023] In some embodiments of the present invention, the compound is obtained by chemical modification based on Michler's Hydrol Blue (MHB). According to literature reports, MHB is highly unstable in aqueous solution with pH>5. This is because the positively charged structure of MHB is very lacking in electricity and is easily attacked by electron-rich nucleophiles, which leads to deterioration. Therefore, the present invention is based on the MHB structure, and the benzene ring of MHB is replaced with a thiophene ring with a high electron cloud density to obtain the compound, thereby improving stability.

[0024] The compound TiCa provided by the present invention can stably exist in an aqueous solution at a physiological pH and a physiological temperature for a long time, and its stability is significantly stronger than that of the compound MHB under the same conditions.

[0025] The compound TiCa provided by the present invention has a long conjugated system in its structure and there are rotatable chemical bonds in the probe molecule. When the compound TiCa is combined with a nucleic acid molecule having a specific secondary structure, the rotatable chemical bonds in the molecule are restricted, which enhances the radiation relaxation process and weakens the non-radiation relaxation process, thereby increasing the fluorescence intensity. This process is also accompanied by a red shift in the fluorescence emission spectrum of the compound.

[0026] The TiCa provided by the present invention has a positive charge in its structure. After entering living cells, it is preferentially enriched in mitochondria with high negative membrane potential, and then binds to negatively charged nucleic acid molecules in mitochondria. For RNA-DNA hybrid nucleic acid molecules with relatively special secondary structures, the compound TiCa provided by the present invention has a high binding ability and response performance at extremely low concentrations. After treating cells with TiCa at a concentration of nanomolar, the TiCa signal observed under a structured light super-resolution microscope is mainly the RNA-DNA hybrid in the mitochondria.

[0027] The second aspect of the present invention provides a method for preparing the compound of the first aspect of the present invention, comprising the following steps:

[0028] The reaction raw materials and methyl trifluoromethanesulfonate are reacted in an organic solvent to obtain;

[0029] The reaction raw materials include At least one of;

[0030] Wherein, R1 and R2 are each independently selected from an amino group or an amine substituent.

[0031] Preferably, R1 and R2 are the same group.

[0032] Preferably, R1 is dimethylamino.

[0033] Preferably, R2 is dimethylamino.

[0034] Preferably, the organic solvent is an alcohol solution.

[0035] Preferably, the reaction temperature is 16-37°C.

[0036] Preferably, after the reaction is completed, the steps of washing, separation and drying are also included.

[0037] In some implementation methods of the present invention, firstly, 5-dimethylaminothiophene-2-carboxaldehyde is used as the reaction raw material, and ethanol is used as the solvent. The reaction reagent methyl trifluoromethanesulfonate (MeOTf) is directly added under stirring at room temperature, and stirring is continued until a gray-purple solid is precipitated and no longer increases. The reaction is stopped. Finally, by suction filtration, washing with ice ethanol and vacuum drying, a gray-purple powder solid with high purity, i.e., the target compound, is obtained. TiCa The preparation reaction formula is as follows:

[0038]

[0039] The third aspect of the present invention provides the first aspect of the present invention Compound Application in the preparation and detection of nucleic acid molecule products.

[0040] Preferably, the product comprises at least one of a probe, a reagent, and a kit.

[0041] Preferably, the nucleic acid molecule has a secondary structure.

[0042] Preferably, the secondary structure comprises at least one of a double helix, a G-quadruplex, a hairpin, and a cross loop.

[0043] Preferably, the nucleic acid molecule comprises at least one of DNA, RNA, and a DNA-RNA hybrid.

[0044] Preferably, the product also includes other reagents for detecting nucleic acid molecules.

[0045] A fourth aspect of the present invention provides a fluorescent probe, comprising the fluorescent probe described in the first aspect of the present invention Compound .

[0046] Preferably, the fluorescent probe further comprises a pharmaceutically acceptable modification.

[0047] Preferably, the pharmaceutically acceptable modification includes at least one of a hydrophilic group, a lipophilic group, and an active group modification.

[0048] Preferably, the pharmaceutically acceptable modification is used to provide corresponding effects for different purposes. For example, improving stability, hydrophilicity, lipophilicity, permeability, low toxicity, etc. Including but not limited to: 1. Isostere modification: the sulfur atom on the aromatic ring can be replaced by an oxygen atom, a selenium atom, etc. 2. Lipophilicity improvement: the aromatic ring part can also introduce some substituents such as halogen atoms, etc., or further modified into a larger conjugated fused heterocycle, such as benzothiophene, etc.

[0049] The fifth aspect of the present invention provides a biosensor, comprising the compound described in the first aspect of the present invention, and / or the fluorescent probe described in the fourth aspect of the present invention, and a primer sequence.

[0050] Preferably, the compound described in the first aspect of the present invention and / or the fluorescent probe described in the fourth aspect of the present invention are modified on the primer sequence.

[0051] In the fourth aspect and the fifth aspect of the present invention, the fluorescent probe and biosensor are used to detect changes in the structure of nucleic acid molecules, or related technical solutions, including a combination of multiple technical solutions, such as probes for detecting RNA-DNA hybrid molecules, biosensors for recognizing DNA complexes in vitro, and can even be used for gel fluorescence imaging after staining of nucleic acid electrophoresis.

[0052] The compound TiCa provided by the present invention can be combined with other technologies in the field to form a probe or biosensor based on different experimental purposes, and achieve corresponding detection and screening purposes.

[0053] The sixth aspect of the present invention provides a kit, comprising the compound described in the first aspect of the present invention, the fluorescent probe described in the fourth aspect of the present invention, and / or the biosensor described in the fifth aspect of the present invention.

[0054] Preferably, the kit further comprises a buffer.

[0055] Preferably, the buffer comprises at least one of TBS, PBS, HBSS and HEPES.

[0056] Preferably, the kit further comprises an instruction manual, which records instructions for use of the kit.

[0057] The seventh aspect of the present invention provides the use of the compound described in the first aspect of the present invention, the fluorescent probe described in the fourth aspect of the present invention, and / or the biosensor described in the fifth aspect of the present invention in the preparation of a product, wherein the product is used for identifying and screening drugs.

[0058] Preferably, the drug functions by affecting nucleic acid secondary structure.

[0059] Preferably, the drug comprises an enzyme, an activator, or an inhibitor.

[0060] In some embodiments of the present invention, the application is the application in detecting mitochondrial RNA-DNA hybrids in living cells.

[0061] In the seventh aspect of the present invention, the application is to utilize the luminescent property of the compound in the secondary structure of nucleic acid to screen drugs that affect the fluorescence intensity.

[0062] Including but not limited to: In the study of mitochondrial DNA and RNA functions, the target DNA and RNA sequences are combined with TiCa. When the target sequence binds to the target sequence and forms a secondary structure, a fluorescent signal is generated. Based on this idea, the functional relationship between RNA and DNA in mitochondria can be studied.

[0063] Including but not limited to: screening drugs that affect nucleic acid structure, or related enzymes. Taking the screening of DNA helicase as an example, a TiCa probe is modified on the DNA double helix molecule, and a candidate enzyme is added. If the candidate enzyme has the unwinding function, it will open the DNA double helix, the DNA molecule loses its secondary structure, and the fluorescence disappears. At this time, the candidate enzyme is judged to be a DNA helicase. If the candidate enzyme does not have the unwinding function, the DNA molecule still has a secondary structure, and the fluorescence is retained. At this time, the candidate enzyme is judged to be a non-DNA helicase. Furthermore, a positive control can be introduced to qualitatively judge the strength of the helicase function based on the change in fluorescence intensity.

[0064] Including but not limited to: cancer-related drug development, for example, DNA G-quadruplexes can regulate genome stability, DNA replication and gene expression in some cancers, and become a therapeutic target for some cancers (such as breast cancer, https: / / www.nature.com / articles / s41588-020-0672-8). TiCa can be combined with DNA G-4 chains to form a composite biosensor to screen candidate drugs. If the candidate drug destroys the DNA G-4 chain, the fluorescence disappears. At this time, it is judged that the drug has the function of destroying the DNA G-4 chain, and its effect in cancer treatment can be further tested. It also includes screening transcription-related inhibitors. For example, when the mitochondrial transcriptase POLRMT operates normally, it can produce an RNA-DNA hybrid structure (or RNA material after transcription is completed). At this time, TiCa will produce fluorescence; if the transcription process is inhibited or promoted by the candidate drug (or biological regulatory factor), the fluorescence signal of TiCa will be weakened or enhanced.

[0065] The eighth aspect of the present invention provides a method for detecting nucleic acid.

[0066] The compound described in the first aspect of the present invention, the fluorescent probe described in the fourth aspect of the present invention, and / or the biosensor described in the fifth aspect of the present invention are used to treat the sample and detect the result.

[0067] In some embodiments of the present invention, the steps of detecting nucleic acid molecules by the compound are as follows: the fluorescent probe is configured into a fluorescent probe solution using the required nucleic acid buffer, which is contacted or mixed with the nucleic acid molecules having a certain concentration, and then obvious orange-red fluorescence or a fluorescent signal near 609nm can be observed under a 560nm fluorescent lamp or a fluorescence detector.

[0068] In some embodiments of the present invention, a method for detecting mitochondrial RNA-DNA hybrids in living cells is provided, the steps of which are as follows: compound TiCa is used to prepare a fluorescent probe solution with a cell culture medium, and then the solution is added to a culture dish containing living cells for treatment, and after the treatment, the living cells are washed with a culture medium, and the distribution of RNA-DNA hybrids in the mitochondria of the living cells can be observed under a structured light super-resolution microscope. More preferably, the concentration of the fluorescent probe used is 2.5 to 10 nM.

[0069] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0070] The compound provided by the present invention belongs to the diarylmethane fluorescent probe, has higher biological stability than the diarylmethane fluorescent probes reported so far, and can be applied to a wider range of biomedical fields. At the same time, the fluorescent probe provided by the present invention has the ability to identify and detect nucleic acid biomacromolecules, and presents a high signal-to-noise ratio detection performance. In terms of the application of living cell imaging, the fluorescent probe TiCa provided by the present invention can detect RNA-DNA hybrid nucleic acid molecules in mitochondria at an extremely low probe concentration. In addition, the preparation process of the fluorescent probe is simple, the cost is low, and the structure is stable and easy to store, which greatly promotes the development of new nucleic acid fluorescent probes and has broad applications in the study of mitochondrial biological functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 This is the NMR characterization of the fluorescent probe TiCa of the present invention. 1 H-NMR spectrum.

[0072] Figure 2 This is the NMR characterization of the fluorescent probe TiCa of the present invention. 13 C-NMR spectrum.

[0073] Figure 3 The three-dimensional structure characterization, X-ray single crystal diffraction pattern and crystal parameters of the fluorescent probe TiCa of the present invention.

[0074] Figure 4The graph is a graph showing the absorbance changes of the fluorescent probe TiCa of the present invention and the reference compound MHB in aqueous solution at physiological pH and physiological temperature for a long time.

[0075] Figure 5 These are the visible light images and fluorescence images of the fluorescent probe TiCa of the present invention before and after binding to nucleic acid, wherein the left image is the visible light image, the middle image is the fluorescence image, and the right image is the visualization result.

[0076] Figure 6 The present invention is a fluorescence titration curve of the fluorescent probe TiCa of the present invention when different types of nucleic acid samples are added dropwise in a Tris-HCl buffer solution; wherein the concentration of the fluorescent probe is 5 nM.

[0077] Figure 7 This is a structured light illumination super-resolution imaging image of living cells after co-staining the fluorescent probe TiCa of the present invention with the commercially available mitochondrial probe MitoTracker Deep Red; wherein, the TiCa probe concentration is 5 nM, the left picture is the TiCa probe labeling result, the middle picture is the MitoTracker labeling result, and the right picture is the Merge result. DETAILED DESCRIPTION

[0078] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0079] Example 1 Preparation and characterization of diarylmethane fluorescent probes

[0080] 1. Preparation of diarylmethane fluorescent probes

[0081] Weigh 150 mg of 5-dimethylaminothiophene-2-carboxaldehyde and dissolve it in 2 mL of ethanol, then slowly drop 240 mg of methyl trifluoromethanesulfonate, and react at room temperature for 24 hours after thorough mixing to obtain a gray-purple suspension. Add 0.5 mL of anhydrous ether to the reaction system, mix thoroughly, let stand, filter, and wash with -20°C ethanol, and dry to obtain 61 mg of gray-purple powder solid, which is the target product TiCa, with a yield of 33%.

[0082] 2. Hydrogen spectrum and carbon spectrum characterization

[0083] The structural formula and data of the target product TiCa are as follows:

[0084]

[0085] 1H NMR (400MHz, DMSO-d6) δ8.07 (s, 1H), 7.82 (d, J = 5.1Hz, 2H), 6.72 (d, J = 5.1Hz, 2H), 3.34 (s, 12H). 13 C NMR (126MHz, DMSO-d6) δ174.04(2C), 148.28(2C), 135.82, 122.41(2C), 111.68(2C), 43.26(4C). Figure 1 is the NMR hydrogen spectrum of TiCa, Figure 2 This is the NMR carbon spectrum of TiCa.

[0086] 3. Crystal parameters

[0087] Take 20 mg of TiCa powder solid, dissolve TiCa completely with 10 mL of methanol: dichloromethane (1:10) mixed solvent, then add 1 mL of ethanol and mix thoroughly, cover the bottle mouth with a paper towel and place it in a ventilated place at room temperature to evaporate slowly, and blue-purple needle-shaped crystals precipitate in the solution after 24 hours. The crystal chemical data of TiCa single crystal is obtained by X-ray single crystal diffraction.

[0088] C 14 H 17 F3N2O3S3 (M=414.47g / mol): monoclinic, space group P21 / c (no.14)

[0089] β=101.006(2)°,Z=4,T=100.00(10)K,μ(Cu K α )=4.393mm -1 ,D calc =1.597g / cm 3 ,11307reflections measured(7.938°≤2Θ≤150.138°),3371unique(R int =0.0318,R sigma =0.0306)which were used in all calculations.The final R1 was0.0798(I>2σ(I))and wR2 was 0.2093.

[0090] Table 1 Figure 3 X-ray single crystal diffraction pattern and crystal parameters of TiCa.

[0091] Table 1

[0092]

[0093] Example 2 Experimental evaluation of the stability of aqueous solution of fluorescent probe TiCa at physiological pH and physiological temperature

[0094] The reference compound Michler's Hydrol Blue (MHB) (synthesized with reference to the relevant literature "Synthesis, Structure, and Properties of Amino-Substituted Benzhydrylium Ions–A Linkbetween Ordinary Carbocations and Neutral Electrophiles" https: / / doi.org / 10.1002 / ejoc.201800835) and the fluorescent probe TiCa were prepared into 5 mL of 10 μM test solution with 1× PBS buffer solution, and placed in a constant temperature box at 37°C in the dark, and the maximum absorption wavelength λ of the corresponding substance was measured after 0 min, 10 min, 30 min, 1 h, 3 h, 5 h, 18 h, 24 h and 48 h. max The absorbance A is plotted, and the standardized A–t curve is plotted ( Figure 4 The results showed that the reference compound MHB completely faded and deteriorated within only 10 minutes, while the TiCa fluorescent probe remained unchanged within 48 hours, indicating that the TiCa fluorescent probe has high stability in aqueous solution at physiological pH and physiological temperature.

[0095] Example 3 Fluorescent probe TiCa responds to nucleic acid biomacromolecules in vitro

[0096] 3 mL of 40 μM probe solution of TiCa was prepared with 1× PBS buffer and placed in two cuvettes respectively. 40 μg of RNA nucleic acid molecules (total RNA extracted from HeLa cells) were added to one of the cuvettes and mixed evenly. Subsequently, the TiCa solution without and with nucleic acid was photographed under white light, and the fluorescence image was obtained using a fully automatic chemiluminescence imager ( Figure 5). It can be observed that TiCa in aqueous solution is bright purple, and when RNA nucleic acid is added, its color instantly changes to dark blue or blue-purple. Under fluorescent light, this blue-purple liquid can emit super strong deep orange-red fluorescence, while there is no obvious fluorescence for the purple solution without RNA added. The corresponding fluorescence spectrum shows that TiCa will produce strong fluorescence at 609nm after binding to nucleic acid, while TiCa itself is only detected with weak fluorescence. Overall, it shows that the TiCa compound is a fluorescent on-off nucleic acid fluorescent probe and a new type of nucleic acid fluorescent probe with a high signal-to-noise ratio, which provides an important basis for subsequent application research at the cellular level.

[0097] Example 4 In vitro recognition of different types of nucleic acid molecules by fluorescent probe TiCa

[0098] The sequences of different types of nucleic acid samples tested are shown in Table 2.

[0099] Table 2

[0100]

[0101]

[0102] Dissolve the corresponding nucleic acid chain in a commercially available 1×TE buffer (pH=8.0) in an appropriate amount, then use an ultra-micro UV to determine the concentration, heat at 95°C for 10 min, then slowly cool and anneal to room temperature as a storage solution, and store at 4°C for later use.

[0103] The test probe TiCa was dissolved in dimethyl sulfoxide to prepare a 1 mM stock solution, which was then diluted with 1×TE buffer (pH=8.0) to prepare 5 μM and 5 nM fluorescent probe solutions for testing.

[0104] The above four different nucleic acid sequence test solutions were added dropwise to the buffer solution of 5 nM fluorescent probe TiCa. As the nucleic acid concentration increased, the change in the fluorescence intensity of the mixed solution at the maximum fluorescence emission point was recorded. The results are as follows: Figure 6 shown.

[0105] The results showed that at the same probe concentration, the probe showed a stronger response to RNA-DNA hybrid double strands and double-stranded DNA with secondary structure, which was significantly higher than single strands. Compared with double-stranded DNA with classic B-type double helix, probe TiCa showed stronger response and binding ability to RNA-DNA hybrid double strands with non-classical double helix, and with the increase of nucleic acid concentration, the fluorescence gain exceeded 120 times. It proves that the fluorescent probe TiCa has great potential to identify the double-stranded structure of RNA-DNA hybrids.

[0106] Example 5 In vitro recognition of different types of nucleic acid molecules by fluorescent probe TiCa

[0107] Human cervical cancer cells HeLa cells were placed in a culture medium (DMEM culture medium and 10v / v% fetal bovine serum) and cultured in an incubator at 37°C, 5% CO2 and 20% O2 for 24 hours. Fluorescent probes TiCa (diluted to a final concentration of 5nM with HeLa cell culture medium) and MitoTracker Deep Red (Invitrogen, M22426, diluted to a final concentration of 50nM with HeLa cell culture medium) were added to the culture dish and cultured for 1 hour. The samples were then washed three times with culture medium and subjected to structured light illumination super-resolution microscopy imaging. The results are shown in Figure 7 .

[0108] The laser channel of MitoTracker is the 647 nm channel (λ ex =640nm,λ em =660–750nm), the hollow rod-shaped fluorescence signal is the signal of the mitochondrial inner membrane; and the laser channel of TiCa is the channel of 561 (λ ex =561nm,λ em =570–670nm), and the point-like fluorescent signal is the distribution position of the mitochondrial RNA-DNA hybrid. It can be observed that all the point-like signals of TiCa are located inside the mitochondria. These results show that the fluorescent probe TiCa provided by the present invention can realize the imaging of RNA-DNA hybrids of mitochondria of living cells at a concentration of 5nM.

Claims

1. A compound, characterized in that: The structural formula of the compound is shown in formula (I): Wherein, R1 and R2 are each independently selected from an amino group or an amine substituent; A - is an anion selected from I - , ClO4 - , TfO - 、BF4 - 、SbF6 - PF6 - , Ts - or FSO2O - .

2. The compound according to claim 1, characterized in that: The amino or amine substituent is selected from at least one of dimethylamino, diethylamino, pyrrolidin-1-yl, piperidin-1-yl, diphenylamino, and azetidin-1-yl.

3. The compound according to claim 2, characterized in that: The structural formula of the compound is shown in formula (II):

4. The method for preparing the compound according to claim 3, comprising the following steps: The reaction raw materials and methyl trifluoromethanesulfonate are reacted in an organic solvent to obtain a compound having a structure represented by formula (I); The reaction raw materials include 5-dimethylaminothiophene-2-carboxaldehyde; Preferably, the organic solvent is an alcohol solution; Preferably, the reaction temperature is 16-37°C; Preferably, after the reaction is completed, the steps of washing, separation and drying are also included.

5. Use of the compound according to any one of claims 1 to 3 in the preparation of a nucleic acid molecule detection product; Preferably, the product comprises at least one of a probe, a reagent, and a kit; Preferably, the nucleic acid molecule has a secondary structure; Preferably, the secondary structure comprises at least one of a double helix, a G-quadruplex, a hairpin, and a cross loop; Preferably, the nucleic acid molecule comprises an RNA-DNA hybrid.

6. A fluorescent probe comprising the compound according to any one of claims 1 to 3; Preferably, the fluorescent probe further comprises a pharmaceutically acceptable modification.

7. A biosensor comprising a primer sequence, and the compound according to any one of claims 1 to 3 and / or the fluorescent probe according to claim 6; Preferably, the compound according to any one of claims 1 to 3 and / or the fluorescent probe according to claim 6 is modified on the primer sequence.

8. A kit comprising the compound according to any one of claims 1 to 3, or the fluorescent probe according to claim 6, or the biosensor according to claim 7; Preferably, the kit further comprises a buffer; Preferably, the buffer comprises at least one of TBS, PBS, HBSS and HEPES.

9. Use of the compound according to any one of claims 1 to 3, or the fluorescent probe according to claim 6, or the biosensor according to claim 7 in the preparation of a product; The products are used to identify and / or screen drugs.

10. A method for detecting a nucleic acid molecule, comprising the following steps: A sample is treated using the compound according to any one of claims 1 to 3, the fluorescent probe according to claim 6, or the biosensor according to claim 7, and the result is detected.

Images