Fluorescent Dye for RNA Detection and Method for Using the Same

Abstract

We provide a rapid and highly accurate virus detection method using RNA-selective fluorescent dyes. [Solution] The method includes the steps of contacting a sample suspected of containing virus particles with a fluorescent probe that is cell membrane permeable and selective for intracellular RNA and a fluorescent dye that binds to the envelope of an enveloped virus, and irradiating the sample with light after the contact; if both a fluorescent signal derived from the fluorescent probe and a fluorescent signal derived from the fluorescent dye that binds to the virus envelope are detected, this indicates a high possibility that the enveloped virus particles are present in the sample.

Description

Technical Field

[0001] The present invention relates to a method for detecting infection with a virus having an envelope and a kit therefor.

Background Art

[0002] As the diverse biological functions of RNA have been revealed, interest in intracellular RNA imaging analysis has been increasing. In particular, fluorescence probes based on small molecules having nucleic acid-binding properties are highly operable and extremely useful. However, in general, most nucleic acid-binding small molecules show higher selectivity for DNA than for RNA, and the development of fluorescence probes having RNA selectivity is not easy.

[0003] As far as the inventor knows, there is only one type of commercially available RNA-selective fluorescence probe, SYTO (trademark) RNAselect (trademark) (Molecular Probes). This shows a green fluorescence response when binding to RNA, and this response is larger compared to the case of binding to DNA. However, the fluorescence wavelength is in the green region, which is a biological background fluorescence region, and it has been pointed out as a problem that the photostability is poor. In addition, this molecule has cell membrane permeability, can stain intracellular RNA, particularly ribosomal RNA in the nucleolus, and is applicable to imaging analysis, but cannot selectively stain the nucleolus in living cells.

[0004] Recently, Nucleolus Bright Green and Nucleolus Bright Red (DOJINDO) have started to be commercially available as nucleolar RNA staining agents. Similar to SYTO RNA select, their compatibility with living cells is low.

[0005] The inventors have previously found that a monomethine cyanine dye (BIQ) composed of a benzo[c,d]indole ring and a quinoline ring has cell membrane permeability and a red fluorescence wavelength (λ em= 657 nm), and has a function of significantly improving the photo-stability, and has been found to be extremely useful for live cell RNA imaging (Non-Patent Document 1). However, a problem with BIQ is that the fluorescence quantum yield (φ fl ) is as low as 0.0085.

[0006] [Chemical Formula]

[0007] In addition, since many viruses in the environment contain RNA in their envelope structures, a fluorescent probe with cell membrane permeability and intracellular RNA selectivity has the potential to rapidly detect and visualize viruses in the environment. The present inventor has developed a highly sensitive RNA-selective staining agent and has studied a more rapid and accurate virus detection method using the RNA-selective staining agent. [Prior Art Documents] [Non-Patent Documents]

[0008] [Non-Patent Document 1] Anal. Chem. 2019, 91, 14254-14260 [Summary of the Invention] [Problems to be Solved by the Invention]

[0009] The problem to be solved by the present invention is to provide a rapid and accurate virus detection method using an RNA-selective fluorescent dye. [Means for Solving the Problems]

[0010] According to a first aspect of the present invention, there is provided a step of contacting a sample suspected of containing virus particles with a fluorescent probe having cell membrane permeability and intracellular RNA selectivity and a fluorescent dye that binds to the envelope of a virus having an envelope, and a step of irradiating the sample with light after the contact. When both a fluorescent signal derived from the fluorescent probe and a fluorescent signal derived from the fluorescent dye that binds to the envelope of the virus are detected, it indicates a high possibility that virus particles having the envelope are present in the sample.

[0011] Also provided is a kit for detecting infection with a virus having an envelope, the kit comprising a fluorescent probe having cell membrane permeability and intracellular RNA selectivity and a fluorescent dye that binds to the envelope of the virus having the envelope.

Advantages of the Invention

[0012] According to a compound according to one aspect of the present invention, RNA can be detected selectively and with high sensitivity. A fluorescent dye according to one aspect of the present invention is applicable to living cells and has clear response performance, and thus can be advantageously used for detecting RNA in a sample. Further, according to a method for detecting virus infection using an RNA-selective fluorescent dye, viruses can be detected more selectively and with high sensitivity.

Brief Description of the Drawings

[0013]

Figure 1

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Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Mode for Carrying Out the Invention

[0014] In a first aspect of the present invention, a compound of the following formula (1) is provided:

[0015]

Chemical formula

[0016] In formula (1), ring A is

Chemical formula

[0017] In the present specification, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11When the moiety of the group can be replaced, the term "replaced" means that one or more (e.g., 1, 2, 3, 4, 5 or 6; in some embodiments 1, 2 or 3; in other embodiments 1 or 2) hydrogens of the group represented by the expression "replaced" can be replaced by a group selected from the listed represented groups or a suitable group known to those skilled in the art, provided that the replacement results in a stable compound. Suitable substituents for the group to be replaced include alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acetylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl, heterocyclesulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl(alkyl)amine and cyano.

[0018] The term "halo" refers to a fluoro group, a chloro group, a bromo group and an iodo group.

[0019] The term "alkyl" refers to a branched, unbranched or cyclic saturated hydrocarbon. In some embodiments, the alkyl group may have, for example, 1 to 20 carbon atoms, often 1 to 12 carbon atoms or 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl (t-butyl), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl and the like. The alkyl may be unsubstituted or substituted. The substituted alkyl group may contain one or more non-carbon and non-hydrogen atoms such as oxygen, nitrogen, sulfur, halogen and phosphorus.

[0020] The term "alkenyl" refers to a branched or unbranched unsaturated hydrocarbon having a carbon-carbon sp 2 double bond. In some embodiments, the alkenyl group can have, for example, 2 to 10 carbon atoms or 2 to 6 carbon atoms. In other embodiments, the alkenyl group has 2 to 4 carbon atoms. Examples of alkenyl include, but are not limited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl and the like. The alkenyl may be unsubstituted or substituted. The substituted alkenyl group may contain one or more non-carbon and non-hydrogen atoms such as oxygen, nitrogen, sulfur, halogen and phosphorus.

[0021] The term "alkynyl" refers to a branched or unbranched unsaturated hydrocarbon chain having a carbon-carbon sp triple bond. In some embodiments, the alkynyl group can have, for example, from 2 to 10 carbon atoms or from 2 to 6 carbon atoms. In other embodiments, the alkynyl group can have from 2 to 4 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1-octynyl, and the like. Alkynyl may or may not be substituted. The substituted alkynyl group may contain one or more non-carbon and non-hydrogen atoms such as oxygen, nitrogen, sulfur, halogen, and phosphorus.

[0022] The term "alkoxy" refers to alkyl-O- (alkyl as defined herein). In some embodiments, the alkoxy group has from 1 to 12 carbon atoms or from 1 to 6 carbon atoms. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, 1,2-dimethylbutoxy, and the like. Alkoxy may or may not be substituted. The substituted alkoxy group may contain oxygen bonded to a substituted alkyl group.

[0023] The term "aryl" refers to an aromatic hydrocarbon group derived by removing one hydrogen atom from one carbon atom of the parent aromatic ring. The aryl group can have 6 to 18 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. The aryl group can have a monocyclic ring (e.g., phenyl) or a polycondensed ring (fused ring) in which at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Typical aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, etc. Aryl may or may not be substituted. For example, the aryl group can be substituted with one or more substituents (as described above) to give various substituted aryls, such as pentafluorophenyl or p-trifluoromethylphenyl, etc.

[0024] The term "amino" refers to -NH 2 . The amino group can be optionally substituted as defined for the term "substituted". For example, the amino group can be -NR 2 (where R is a group listed in the definition of "substituted"). For example, the group -NR 2 can include "alkylamino" (at least one R is alkyl and the other R is alkyl or hydrogen) and / or "acylamino" (-N(R)C(=O)R) (each R is independently hydrogen, alkyl, alkaryl, or aryl). The amino group can be a primary amine (NH 2 ), a secondary amine (NHR), or a tertiary amine.

[0025] Since the fluorescence quantum yield φ fl of the monomethine cyanine dye BIQ is extremely low, the inventors attempted to design and synthesize new cyanine dyes based on the structures of some known fluorescent dyes with high fluorescence quantum yields φ fl , and designed and synthesized a new monomethine cyanine dye in which a benzo[c,d]indole ring and an oxalopyridine ring are linked.

[0026] Since the compound of the above formula (1) emits fluorescence upon binding to nucleic acids, it can be used as a fluorescent dye. Further, the compound of the above formula (1) is selective for RNA over DNA and can detect RNA with high sensitivity. The fluorescent dye containing the compound of the above formula (1) or the fluorescent dye consisting of the compound of the above formula (1) is applicable to living cells and can selectively stain nucleosomes rich in RNA at a low concentration (1.0 μM) for a short time (20 min) and has a clear response performance, so it can be preferably used for intracellular RNA imaging.

[0027] The compound of the above formula (1) may be covalently bonded to other molecules such as antibodies, proteins, peptides, polypeptides, amino acids, enzymes, nucleic acids, lipids, polysaccharides, drugs, beads, solid supports (e.g., glass or plastic).

[0028] Preferably, the compound of the above formula (1) emits little or no fluorescence in the absence of nucleic acids. Fluorescence can be measured by irradiating the compound with an appropriate wavelength and monitoring the emitted fluorescence. Preferably, the compound emits stronger fluorescence in the presence of RNA than in the presence of DNA. The fluorescence in the presence of RNA relative to the fluorescence in the presence of DNA is measured with the compound concentration kept constant and the RNA and DNA concentrations kept constant. The higher the RNA / DNA fluorescence ratio, the more suitable it is for RNA detection in the presence of DNA. The RNA / DNA fluorescence ratio is preferably 1 or more, more preferably greater than 1, 1.2 or more, 1.5 or more, and 2 or more. The compound of the above formula (1) meets the criteria suitable for RNA detection.

[0029] The compound of formula (1) can also be characterized by its maximum excitation and emission wavelengths. For example, the maximum excitation may be from about 450 nm to about 650 nm. The maximum excitation between these values may be about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, and between any two of these values. For example, the maximum emission may be from about 500 nm to about 675 nm. The maximum emission between these values may be about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, and between any two of these values.

[0030] In a preferred embodiment, the compound of formula (1) is a compound of formula (1a) or formula (1b).

Chemical formula

[0031] In some embodiments of formula (1), (1a), and (1b), R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are independently hydrogen, a hydroxy group, a thiol group, a halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, an amino group, and R 5 is an alkyl group.

[0032] In some embodiments of formula (1), (1a), and (1b), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11is independently hydrogen, a hydroxy group, halo, an alkyl group, an aryl group, or an amino group.

[0033] In some embodiments of Formulas (1), (1a), and (1b), R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are independently hydrogen, a hydroxy group, a thiol group, halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group, all of which are unsubstituted, and R 5 is also unsubstituted. In some preferred embodiments, R 1 , R 2 , R 3 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are hydrogen, R 4 is alkyl having 1 to 6 carbon atoms, and R 5 is methyl.

[0034] In some preferred embodiments, the compound of Formula (1) is a compound of Formula (2a). Since the compound of Formula (2a) is a combination of Benzo[c,d]Indole and OxazoloPyridine, it may hereinafter also be referred to as BIOP.

[0035]

Chemical Formula

[0036] The compound of formula (2a) is selective for RNA over DNA and can detect RNA with high sensitivity. Also, it has a high fluorescence intensity change and higher photo-stability than known RNA probes such as SYTO RNA select (trademark) (Invitrogen). Furthermore, the fluorescent dye composed of the compound of formula (2a) is applicable to living cells, and can selectively stain nucleosomes rich in RNA at a low concentration (1.0 μM) for a short time (20 min), and has a clear response performance, so it can be suitably used for intracellular RNA imaging.

[0037] Taking the case where the compound of formula (2a) (BIOP) uses Benzo[c,d]indole-2(1H)-one (1) (benzo[c,d]indol-2(1H)-one (1)) as a starting material as an example, it can be produced according to the following Scheme 1.

[0038]

Chemical formula

[0039] First, using Benzo[c,d]indole-2(1H)-one (Compound 1), 1-methylbenzo[c,d]indol-2(1H)-one (Compound 2) is obtained according to J. Med. Chem., 2016, 59, 1565-1579. A 1,4-dioxane solution containing the obtained Compound 2 (0.55 g, 3.0 mmol) and Lawesson's reagent (2.43 g, 6.0 mmol) is stirred at 100 °C overnight. This solution is filtered at room temperature, and the obtained solid is washed with 1,4-dioxane to obtain the 1-methylbenzo[c,d]indol-2(1H)-thione crude product 3. Next, methyl iodide is added to the crude product 3 (1.71 g), and the mixture is heated to reflux overnight. After concentrating this solution under reduced pressure, diethyl ether is added, and the mixture is sonicated for 30 minutes. The obtained solid is collected by filtration to obtain 1-methyl-2(methylthio)benzo[c,d]indole-1-ium (Compound 4). Next, 0.5 mL of tetraethylamine is added to an acetonitrile solution containing 4-methyl-2-methyl-oxazolo[4,5]pyridinium iodide (Compound 5, 193 mg, 0.70 mmol) and Compound 4 (162 mg, 0.47 mmol), which is obtained by the reaction of 2-methyl-oxazolo[4,5-b]pyridine and methyl iodide. This solution is reacted at reflux (60 °C) for 1 hour while stirring, and then cooled to room temperature. Thereafter, diethyl ether is added, and the obtained precipitate is collected by filtration. Thereafter, water is added, and the product is collected by filtration again and dried to obtain Compound 6 (BIOP).

[0040] In some preferred embodiments, the compound of formula (1) is a compound of formula (2b). The compound of formula (2b) may also be referred to as BIOP[5,4-c].

[0041]

Chemical formula

[0042] The compound of formula (2b) is selective for RNA over DNA and can detect RNA with high sensitivity. In addition, it has a high fluorescence intensity change and higher photostability than known RNA probes such as SYTO RNA select. The compound of formula (2b) has a significantly improved fluorescence quantum yield compared to the compound of formula (2a). Furthermore, the fluorescent dye composed of the compound of formula (2b) is applicable to living cells and can selectively stain nucleosomes rich in RNA at a low concentration (1.0 μM) for a short time (20 min), and has a clear response performance, so it can be preferably used for intracellular RNA imaging.

[0043] The compound of formula (2b) (BIOP[5,4-c]) can be produced, for example, according to the following Scheme 2. Until compound 4 (1-methyl-2(methylthio)benzo[c,d]indole-1-ium) is obtained, it is the same as that described in the production process of BIOP mentioned above. Using compound 4, according to Dyes and Pigments., 1991, 15, 215-223., through compound 7 ((1-methyl-2(methylthio)benzo[c,d]indole-1-ium)), finally compound 8 (1,2-dimethylbenzo[cd]indol-1-ium iodide) is obtained.

[0044] [Chemical formula]

[0045] On the other hand, using 4-aminopyridin-3-ol as a starting material, according to Eur. J. Med. Chem., 2005, 40, 15-23., compound 9 (2-(methylthio)oxazolo[5,4-c]pyridine) is obtained. According to J. Mater. Chem. B., 2014, 2, 2688-2693., compound 10 (5-methyl-2-(methylthio)oxazolo[5,4-c]pyridin-5-ium tosylate) is obtained from compound 9 (Scheme 3).

[0046]

Chem.

[0047] To an acetonitrile solution containing Compound 8 (110 mg, 0.36 mmol) prepared according to Scheme 2 and Compound 10 (100 mg, 0.23 mmol) prepared according to Scheme 3, 0.8 mL of tetraethylamine is added. After reacting this solution at 40 °C for 22 hours with stirring, it is cooled to room temperature. Then, diethyl ether is added and the resulting precipitate is collected by filtration. After dissolving this precipitate in methanol, 20 mL of a sodium iodide solution is added dropwise. After reacting this solution at room temperature for 2 hours with stirring, the resulting precipitate is collected by filtration and dried to obtain BIOP(5,4-c) (Scheme 4).

[0048]

Chem.

[0049] In some preferred embodiments, the compound of formula (1) is a compound of formula (2c). The compound of formula (2c) may also be referred to as BIOP[5,4-c]-nBu.

[0050]

Chem.

[0051] The compound of formula (2c) can detect RNA with high sensitivity. Also, it has a high fluorescence intensity change and higher photostability than known RNA probes such as SYTO RNA select. The compound of formula (2c) has a significantly improved fluorescence quantum yield compared to the compound of formula (2a). Furthermore, the fluorescent dye composed of the compound of formula (2c) can be applied to living cells and can selectively stain nucleosomes rich in RNA at a low concentration (1.0 μM) for a short time (20 min) and has a clear response performance, so it can be suitably used for intracellular RNA imaging.

[0052] The compound (BIOP[5,4-c]-nBu) of formula (2c) can be produced, for example, according to Scheme 5 below. Until the compound 9 (2-(methylthio)oxazolo[5,4-c]pyridine) is obtained, it is the same as that described in the production process of BIOP[5,4-c] mentioned above. A solution of acetonitrile containing compound 9 (0.20 g, 1.2 mmol) and 1-iodobutane (0.81 g, 4.4 mmol) is reacted under heating to reflux with stirring for 14 hours. Then, the solvent is removed under reduced pressure to obtain the crude product 11 (5-butyl-2-(methylthio)oxazolo[5,4-c]pyridin-5-ium iodide).

[0053]

Chemical formula

[0054] On the other hand, prepare compound 8 (1,2-dimethylbenzo[cd]indol-1-ium iodide) as described in the production process of BIOP[5,4-c]. Add 0.8 mL of tetraethylamine to a solution of acetonitrile containing compound 8 (150 mg, 0.49 mmol) and compound 11 (220 mg, 0.63 mmol) produced in Scheme 5. After reacting this solution at 40 °C with stirring for 17 hours, it is cooled to room temperature. Then, diethyl ether is added and the resulting precipitate is collected by filtration. This precipitate is purified by silica gel chromatography. The obtained crude product is dissolved in methanol, and then water is added dropwise. The resulting precipitate is collected by filtration and dried to obtain BIOP(5,4-c)-nBu (Scheme 6).

[0055]

Chemical formula

[0056] In a second aspect of the present invention, there is provided a method comprising the steps of mixing the compound of formula (1) with a sample containing RNA to form a mixed sample in which the compound is bound to the RNA in the sample, and irradiating the mixed sample to detect the RNA in the mixed sample.

[0057] The details of the compound of formula (1) are as described for the compound of formula (1) in the first aspect.

[0058] The sample containing RNA is not particularly limited. For example, the sample may be one or more types of cells, tissues, cell lysates, cell culture media, etc. Alternatively, the sample may be an abiotic sample. The cells may be any cells such as bacterial cells, fungal cells, insect cells, and mammalian cells. Mammalian cells are preferably human cells. In a preferred embodiment, the cells are live cells. The sample may be solid, liquid or suspension. The sample may be a biological fluid such as blood, plasma or urine. The sample may be fixed in a gel or on a membrane, bound to one or more beads, or configured in the form of an array. The sample may also be a buffer or water containing a partially or wholly purified nucleic acid preparation.

[0059] The step of mixing the compound of formula (1) with a sample containing RNA to form a mixed sample in which the compound is bound to the RNA in the sample may be carried out at any suitable temperature and time. Typically, the temperature is normal temperature or room temperature. Examples of such temperature include about 20°C, about 25°C, about 30°C, about 35°C, about 37°C, about 40°C, about 42°C, and ranges between any two of these values. Temperatures above about 42°C and below about 20°C may also be applicable depending on the test sample. The time is not particularly limited and may be any time suitable for detecting fluorescence changes. Examples of the length of time include about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, and ranges between any two of these values. It is also possible to extend the time depending on the test sample.

[0060] The concentration of the compound of formula (1) is not particularly limited and may be any concentration at which the fluorescence excitation and emission signals can be appropriately detected in the presence of RNA. Examples of the concentration range include about 10 nM to 1 mM. Examples of the concentration include about 10 nM, about 100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, and the ranges between any two of these values.

[0061] Suitable irradiation devices include portable ultraviolet lamps, mercury arc lamps, xenon lamps, lasers (such as argon and YAG lasers), and laser diodes. These irradiation sources are typically optically integrated into a laser scanner, a fluorescence microplate reader, a standard spectrophotometer, or a micro-fluorescence spectrophotometer.

[0062] The detection step may be carried out by visual inspection or by using various measuring instruments. Examples of the measuring instruments include a CCD camera, a video camera, photographic film, a laser scanner device, a fluorescence intensity meter, a photodiode, a quantum counter, an epifluorescence microscope, a scanning microscope, a flow cytometer, a fluorescence microplate reader, or an amplification device such as a photomultiplier tube.

[0063] The detection step may be carried out at a single point in time, at multiple points in time, or continuously.

[0064] In a third aspect of the present invention, there is provided a kit for detecting RNA in a sample, the kit comprising the compound of formula (1) as described above and instructions for detecting RNA in the sample.

[0065] Details of the compound of formula (1) are as described for the compound of formula (1) in the first aspect.

[0066] The sample is not particularly limited as long as it contains RNA. For example, the sample may be one or more types of cells, tissues, cell lysates, cell culture media, etc. Alternatively, the sample may be an abiotic sample. The cells may be any cells such as bacterial cells, fungal cells, insect cells, and mammalian cells. Mammalian cells are preferably human cells. In a preferred embodiment, the cells are live cells. The sample may be solid, liquid, or suspension. The sample may be a biological fluid such as blood, plasma, or urine. The sample may be fixed in a gel or on a membrane, may be bound to one or more beads, or may be configured in the form of an array. The sample may also be a buffer or water containing a partially or wholly purified nucleic acid preparation.

[0067] In a preferred embodiment, the kit comprises a container for containing the compound of formula (1). The kit may also comprise a pipette, dropper, or other sample manipulation substrate.

[0068] The kit may also comprise a positive control and / or a negative control sample. The positive control sample may contain RNA and / or RNA coexisting with DNA. The negative control sample may be a sample containing DNA without RNA, or may be a sample containing no nucleic acid at all.

[0069] The kit may also comprise one or more additional dyes or stains. For example, the kit may comprise a total nucleic acid stain. The kit may also comprise a cell-permeable nucleic acid stain for distinguishing live cells from dead cells.

[0070] The kit may further comprise water, buffer, buffer salts, surfactants, surface-active agents, salts, polysaccharides, or other materials commonly used in bioassays. The kit may also comprise a solvent such as a water-soluble, water-insoluble, or water-soluble / water-insoluble solvent system.

[0071] In a fourth aspect of the present invention, there is provided a method for detecting virus particles having an envelope in a sample, the method including a step of contacting a fluorescent probe composed of the compound of formula (1) or the compound of formula (3) below and a fluorescent probe that binds to the envelope of the virus with a sample in which the presence of virus particles is suspected, and a step of irradiating the sample with light after the contacting. When both of the two types of fluorescent probes exhibit a fluorescence response, it indicates a high possibility that virus particles are present in the sample.

[0072] FIG. 1 schematically shows a method for detecting virus particles in a sample according to the fourth aspect.

[0073] The virus particle (1) has an envelope (2) as an outer coat and virus RNA (3) encapsulated in the envelope (2). The diameter D is about 100 nm. Therefore, when a fluorescent dye as an envelope-binding probe (4) that selectively binds to the envelope (2) of the virus and the compound of formula (1) or the compound of formula (3) below as an RNA-binding probe (5) that selectively binds to the virus RNA (3) are contacted with a sample in which the presence of virus particles (1) is suspected, if virus particles (1) are present in the sample, the envelope (2) and the fluorescent dye bind, and an increase in the luminescence intensity derived from the binding can be detected. At the same time, the virus RNA (3) and the compound of formula (1) or the compound of formula (3) below bind, and an increase in the luminescence intensity derived from the binding can be detected.

[0074] If virus particles (1) are not present in the sample, an increase in the luminescence intensity derived from one or both of the binding of the fluorescent dye to the envelope (2) and the binding of the compound of formula (1) or the compound of formula (3) below to the virus RNA (3) cannot be detected.

[0075] Thereby, the presence or absence of virus particles in the sample can be detected or determined quickly, simply, and with high sensitivity.

[0076] In addition, by using two types of fluorescent dyes, namely a compound of formula (1) that selectively binds to the encapsulated viral RNA or a compound of formula (3) below, and a fluorescent probe that binds to the envelope of the virus, when both dyes bind to the virus particles, fluorescence energy transfer may occur, and it may be possible to distinguish the type and characteristics of the virus by detecting these fluorescence intensities.

[0077] The virus constituting the virus particles may be any virus having RNA as a genome and also having an envelope structure, and examples include, but are not limited to, coronaviruses, influenza viruses, herpesviruses, rubella viruses, hepatitis B and C viruses, and AIDS viruses.

[0078] The details of the compound of formula (1) are as described for the compound of formula (1) in the first aspect.

[0079] The compound of formula (3) is represented as follows.

[0080]

Chemical formula

[0081] In the formula, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、and R 12 are each independently hydrogen, a hydroxy group, a thiol group, a halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.

[0082] In this specification, R 1 、R 2 、R 3 、R 4 、R 5 、R6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 When the base moieties of and R can be substituted, the term "substituted" means that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2, or 3; in other embodiments 1 or 2) hydrogens of the group represented by the expression "substituted" can be substituted with a group selected from the listed indicated groups or a suitable group known to those skilled in the art, provided that the substitution results in a stable compound. Suitable substituents for the substituted group include alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acetylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl, heterocyclesulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl(alkyl)amine and cyano.

[0083] The term "halo" refers to a fluoro group, a chloro group, a bromo group and an iodo group.

[0084] The term "alkyl" refers to a branched, unbranched, or cyclic saturated hydrocarbon. In some embodiments, the alkyl group can have, for example, from 1 to 20 carbon atoms, often from 1 to 12 carbon atoms or from 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl (t-butyl), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. The alkyl may or may not be substituted. The substituted alkyl group may contain one or more non-carbon and non-hydrogen atoms such as oxygen, nitrogen, sulfur, halogen, and phosphorus.

[0085] The term "alkenyl" refers to a branched or unbranched unsaturated hydrocarbon having a carbon-carbon sp 2 double bond. In some embodiments, the alkenyl group can have, for example, from 2 to 10 carbon atoms, or can have from 2 to 6 carbon atoms. In other embodiments, the alkenyl group has from 2 to 4 carbon atoms. Examples of alkenyl include, but are not limited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl, and the like. The alkenyl may or may not be substituted. The substituted alkenyl group may contain one or more non-carbon and non-hydrogen atoms such as oxygen, nitrogen, sulfur, halogen, and phosphorus.

[0086] The term "alkynyl" refers to a branched or unbranched unsaturated hydrocarbon chain having a carbon-carbon sp triple bond. In some embodiments, the alkynyl group can have, for example, 2 to 10 carbon atoms or 2 to 6 carbon atoms. In other embodiments, the alkynyl group can have 2 to 4 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1-octynyl, and the like. Alkynyl may or may not be substituted. The substituted alkynyl group may contain one or more non-carbon and non-hydrogen atoms such as oxygen, nitrogen, sulfur, halogen, and phosphorus.

[0087] The term "alkoxy" refers to alkyl-O- (where alkyl is defined herein). In some embodiments, the alkoxy group has 1 to 12 carbon atoms or 1 to 6 carbon atoms. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, 1,2-dimethylbutoxy, and the like. Alkoxy may or may not be substituted. The substituted alkoxy group may contain oxygen bonded to a substituted alkyl group.

[0088] The term "aryl" refers to an aromatic hydrocarbon group derived by removing one hydrogen atom from one carbon atom of the parent aromatic ring. The aryl group can have 6 to 18 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. The aryl group can have a monocyclic ring (e.g., phenyl) or a polycondensed ring (fused ring) in which at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Typical aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, etc. The aryl may or may not be substituted. For example, the aryl group can be substituted with one or more substituents (as described above) to give various substituted aryls, such as pentafluorophenyl or p-trifluoromethylphenyl.

[0089] The term "amino" refers to -NH 2 . The amino group can be optionally substituted as defined for the term "substituted". For example, the amino group can be -NR 2 (where R is a group listed in the definition of "substituted"). For example, the group -NR 2 can include "alkylamino" (at least one R is alkyl and the other R is alkyl or hydrogen) and / or "acylamino" (-N(R)C(=O)R) (each R is independently hydrogen, alkyl, alkaryl, or aryl). The amino group can be a primary amine (NH 2 ), a secondary amine (NHR), or a tertiary amine.

[0090] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12is independently hydrogen, a hydroxy group, a thiol group, halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group, and R 6 is an alkyl group.

[0091] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 is independently hydrogen, a hydroxy group, halo, an alkyl group, an aryl group, or an amino group.

[0092] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 is independently hydrogen, a hydroxy group, a thiol group, halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group, all of which are unsubstituted, and R 6 is also unsubstituted.

[0093] In some preferred embodiments, the compound of formula (3) is a compound of formula (4) (monomethine cyanine dye (BIQ)).

[0094]

Chemical formula

[0095] The compound of formula (3) can be prepared by the method disclosed in Anal. Chem. 2019, 91, 14254 - 14260 (Scheme 7).

[0096]

Chem.

[0097] Until the crude product of Compound 4 is obtained, it is the same as the production of the compound of formula (1). Thereafter, 0.5 mL of tetraethylamine is added to an acetonitrile solution containing 1,4-dimethylquinolin-1-ium (Compound 12, 258 mg, 0.91 mmol) obtained by the reaction of 4-methylquinoline and iodomethane and the crude product of Compound 4 (194 mg, 0.57 mmol). After reacting this solution under heating under reflux with stirring for 1 hour, it is cooled to room temperature. Thereafter, diethyl ether is added to the solution, and a precipitate can be obtained by filtration. After washing the obtained precipitate with water, it is dissolved in methanol. Thereafter, diethyl ether is added, and the obtained precipitate is dried to obtain Compound 13 (BIQ).

[0098] On the other hand, examples of the fluorescent dye that binds to the envelope of the virus include peptidic fluorescent dyes and cell membrane staining dyes that can selectively bind to the lipid bilayer structure of the envelope.

[0099] As peptide fluorescent dyes that can bind highly selectively to the lipid bilayer structure of the envelope, there are, for example, binding peptides targeting exosomes previously developed by the inventors (The 98th Spring Annual Meeting of the Chemical Society of Japan (2018 (4G3-02)), The 68th Annual Meeting of the Japan Society for Analytical Chemistry (2019 (D1101, D1102, L2006)), RSC Advances, 10, 38323-38327 (2020). [DOI: 10.1039 / d0ra07763a]). This is based on the fact that exosomes have a highly curved lipid bilayer with a diameter of about 100 nm, and a hydrophobic region called lipid packing defect appears locally on the surface of the lipid bilayer. The binding peptide uses an amphiphilic α-helix peptide as a binding motif for the lipid packing defect, and an environmentally responsive fluorescent dye is linked to this. Since viruses have a diameter similar to that of exosomes, it can be expected that such a fluorescent dye based on an α-helix peptide can bind selectively to the envelope of the virus.

[0100] In the design of peptide fluorescent dyes that can bind selectively to the lipid bilayer structure of the envelope, the peptide part is designed to recognize the lipid packing structure of the envelope, and the environmentally responsive fluorescent dye is attached to a position that does not interfere with the binding of the envelope and the peptide, such as the N-terminus or C-terminus of the peptide.

[0101] The design of amphiphilic α-helix peptides that bind to lipid packing defects is described in FEBS Lett., 2010, 584, 1840-1847. Such α-helix peptides include α-helix peptides such as ArfGAPl (R. norvegicus), nucleoporin Nupl33 (H. sapiens), α-synuclein (H. sapiens), DivIVA (B. subtillis), dAmph (D. melanogaster), endophilin Al (R. norvegicus), Sarlp (S. cervisiae), antimicrobial magainin 2 (X. laevis), the string-like N-terminus of GMAP-210 (H. sapiens), sterol transporter Keslp (S. cerevisiae), SpoVM (B. subtilis), Epsin (H. sapiens), Binl / amphiphysin II (H. sapiens), H0-NBAR derived from BRAP / Bin2, myristoylated Arf 1 (B. taurus), melittin (A. mellifera), etc., or peptides based on these α-helix peptides (e.g., a part of an α-helix peptide, 1 to several, more specifically about 1 to 5 amino acids mutated amino acids of an α-helix peptide), but are not limited thereto. Those skilled in the art can appropriately select the sequences of various peptides as the peptide of the peptidic fluorescent dye that can selectively bind to the lipid bilayer structure of the envelope.

[0102] As the environment-responsive fluorescent dye, any dye that has a weak fluorescence intensity in a hydrophilic environment but an increased intensity in a hydrophobic environment can be used, and such dyes are known in the art. Examples of the environment-responsive fluorescent dye include dansyl dyes and their derivatives, Dapoxyl dyes and their derivatives, benzophenoxazine dyes and their derivatives, and the like. Specific examples of the environment-responsive fluorescent dye include allylnaphthalenesulfonic acids such as 1-anilinonaphthalene-8-sulfonic acid (ANS), N-methyl-2-anilinonaphthalene-6-sulfonic acid (MANS), 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS); dimethylaminonaphthalenesulfonic acid; benzofurazan derivatives such as nitrobenzofurazan (NBD); Dapoxyl dyes (Benzenesulfonic acid, 4-[5-[4-(dimethylamino)phenyl]-2-oxazolyl); Dapoxyl derivatives such as Dapoxyl sulfonyl chloride, Dapoxyl succinimidyl ester, Dapoxyl 3-sulfonamidopropionic acid, Dapoxyl (2-bromoacetamidoethyl) sulfonamide, Dapoxyl (2-aminoethyl) sulfonamide; dansyl dyes such as dansyl chloride, dansyl sulfonamide, dansylaminoethyl-3-phosphate, 1-dansylsulfonamide-3-N,N-dimethylaminopropane, dansylcholine, dansylgalactoside, dansyllysine, dansylphosphatidylethanolamine; benzophenoxazine derivatives such as Nile Red and Nile Blue, and the like can be exemplified.

[0103] In accordance with this principle, a peptide-based fluorescent dye that selectively binds to the envelope of a virus and exhibits a fluorescence response can be prepared.

[0104] Preferred embodiments of the peptide-based probe that can selectively bind to the lipid bilayer structure of the envelope of a virus include amphiphilic α-helix peptide probes such as the compound of the following formula (9).

[0105]

Chem.

[0106] The peptide-like probe of the above formula (9) can be produced as follows.

[0107] First, after extending the peptide chain using an amino acid protected with an Fmoc group, it is produced by linking a fluorescent dye Nile Red derivative. Specifically, an Fmoc group-protected amino acid is linked to Rink-Amide-ChemMatrix resin (Biotage) using [1-(1-cyano-ethoxy-2-oxoethylideneamino-oxy)-dimethylamino-morpholino methylene]methanaminium hexafluorophosphate (COMU) and diisopropylethylamine (DIEA). Subsequently, a Nile Red derivative (6-((9-(diethylamino)-5-oxo-5H-benzo[a]phenoxazin-2-yl)oxy)hexanoic acid) synthesized according to J. Chem. Soc., Perkin Trans., 1997, 1, 1051-1058. is linked. Then, after treatment of cleavage from the resin and deprotection, the resulting crude product is purified by reverse-phase HPLC to obtain a peptide-like fluorescent probe.

[0108] As the fluorescent dye used for the peptide probe described above, not only environmentally responsive dyes but also non-environmentally responsive dyes such as fluorescein dyes and their derivatives, and rhodamine dyes and their derivatives can be used. However, the fluorescent probe using an environmentally responsive dye has the characteristics that the fluorescence intensity is small in a hydrophilic field and large in a hydrophobic field, so that fluorescence can be detected without operations such as washing. On the other hand, in the fluorescent probe using a non-environmentally responsive dye, it is impossible to distinguish between the non-bound state and the bound state to the envelope, so operations such as washing are required. Preferably, an environmentally responsive dye such as the compound of formula (9) is preferred, but depending on the purpose and application, the use of a non-environmentally responsive dye may be considered.

[0109] As the cell membrane staining dye, a known dye capable of staining the cell membrane can be used, and in particular, a lipophilic dye that stains the lipid bilayer is preferred. Examples of such lipophilic dyes include lipophilic carbocyanine dyes such as the PIC series of Takara Bio Inc. and SP-DiOC1 8 (3) in the Molecular Probes (registered trademark) series manufactured by Thermo Fisher Scientific, and Polaric (registered trademark) manufactured by Goryo Chemical Co., Ltd. Lipophilic carbocyanine dyes are usually carbocyanine dyes having a long-chain hydrocarbon chain. In addition to these, as the cell membrane staining dye, lipophilic dyes such as Sudan III, Sudan II (Oil Red O), Sudan Black B (SBB), Nile Blue, Fat Red, and Lipid Crimson, which have been conventionally used for lipid staining, can also be used in the same manner.

[0110] The sample containing RNA is not particularly limited. For example, the sample may be one or more types of cells, tissues, cell lysates, cell culture media, etc. Alternatively, the sample may be an abiotic sample. The cells may be any cells such as bacterial cells, fungal cells, insect cells, and mammalian cells. The mammalian cells are preferably human cells. In a preferred embodiment, the cells are live cells. The sample may be solid, liquid or suspension. The sample may be a biological fluid such as blood, plasma or urine. Also, it may be something captured in a state of floating in the air. The sample may be fixed in a gel or on a membrane, may be bound to one or more beads, or may be configured in the form of an array. The sample may be a buffer or water containing a partially or wholly purified nucleic acid preparation.

[0111] The contacting step may be carried out at any appropriate temperature and time. Typically, the temperature is normal temperature or room temperature. Examples of such temperature include about 20°C, about 25°C, about 30°C, about 35°C, about 37°C, about 40°C, about 42°C, and ranges between any two of these values. Temperatures above about 42°C and below about 20°C may also be applicable depending on the test sample. The time is not particularly limited and may be any time appropriate for detecting the fluorescence change. Examples of the length of time include about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, and ranges between any two of these values. Furthermore, it is also possible to extend the time depending on the test sample.

[0112] Also, the medium for contacting may be any kind. A mixture of the compound of formula (1) or the compound of formula (3) and a fluorescent probe that binds to the envelope of the virus may be directly mixed with the sample. Alternatively, for example, a medium such as a mask may be impregnated with a mixture containing the compound of formula (1) or the following compound of formula (3) and a fluorescent probe that binds to the envelope of the virus, and a sample floating in the air may be captured and reacted.

[0113] The concentrations of the compound of formula (1) and the compound of formula (3) are not particularly limited and may be any concentrations at which the fluorescence excitation and emission signals can be appropriately detected in the presence of RNA. Examples of the concentration range include about 10 nM to 1 mM. Examples of the concentrations include about 10 nM, about 100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, and ranges between any two of these values.

[0114] Suitable irradiation devices include portable ultraviolet lamps, mercury arc lamps, xenon lamps, lasers (such as argon and YAG lasers), and laser diodes. These irradiation sources are typically optically integrated into a laser scanner, a fluorescence microplate reader, a standard spectrophotometer, or a micro-fluorescence spectrophotometer.

[0115] The detection or determination step may be carried out by visual inspection or by using various measuring instruments. Examples of the measuring instruments include a CCD camera, a video camera, photographic film, a laser scanner device, a fluorescence intensity meter, a photodiode, a quantum counter, an epi-fluorescence microscope, a scanning microscope, a flow cytometer, a fluorescence microplate reader, or an amplification device such as a photomultiplier tube.

[0116] The detection or determination step may be carried out at a single point in time, at multiple points in time, or continuously.

[0117] When both the fluorescence signal derived from the compound of formula (1) or the following formula (3) and the fluorescence signal derived from the fluorescent probe that binds to the envelope of the virus are detected, it can be detected or determined that there is a high possibility that virus particles are present in the sample.

[0118] In a fifth aspect of the present invention, there is provided a method for detecting virus particles having an envelope in a sample, the method comprising the steps of contacting a fluorescent probe comprising the compound of formula (1) or the compound of formula (3) below with a sample in which the presence of virus particles is suspected, and irradiating the sample with light after the contacting, wherein if the fluorescent probe exhibits a fluorescence response, it indicates a high likelihood of the presence of virus particles in the sample.

[0119] Details of each step and the materials used in the fifth aspect of the present invention are as described in the fourth aspect of the present invention.

[0120] In the method for detecting virus particles according to the fourth aspect of the present invention described above, a fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below and a fluorescence signal derived from a fluorescent probe that binds to the envelope are detected. However, the method for detecting virus particles of the present invention is not limited thereto. As another detection method, for example, in the fifth aspect of the present invention, a fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below is detected. When comparing the RNA in virus particles with the RNA in solution, in the case of virus particles, since it is a spatially limited system, it is observed as a bright spot having a strong intensity. On the other hand, the RNA in solution and the fluorescent probe that binds to the envelope cause the entire solution to glow faintly. Utilizing this, virus particles may be detected from the ratio of bright spots having a strong fluorescence intensity. According to the fourth aspect of the present invention, virus particles can be detected using only the fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below.

[0121] In a sixth aspect of the present invention, there is provided a method for detecting virus infection of cells in vitro, the method comprising the step of contacting the compound of formula (1) or the compound of formula (3) above with a fluorescent dye that binds to the envelope of a virus and a cell in which virus infection is suspected, and the step of irradiating the cell with light after the contacting, When both a fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below and a fluorescence signal derived from a fluorescent probe that binds to the envelope of the virus are detected, a method is provided indicating that the cell is highly likely to be infected with the virus.

[0122] The details of the compound of formula (1) and the compound of formula (3) are as described with respect to the compound of formula (1) in the first aspect and the method in the fourth aspect, and are the same as the fourth aspect except that in vitro, the compound of formula (1) or the compound of formula (3) above is contacted with a fluorescent dye that binds to the envelope of the virus and a cell suspected of being infected with the virus.

[0123] In a further embodiment of the sixth aspect of the present invention, the average value of the fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below in a virus-infected person, the median value of the fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below in a virus-infected person, or the value of the fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below that separates virus-infected persons from non-virus-infected persons is set as the first threshold value. Also, the average value of the fluorescence signal derived from the fluorescent probe that binds to the envelope of the virus in a virus-infected person, the median value of the fluorescence signal derived from the fluorescent probe that binds to the envelope of the virus in a virus-infected person, or the value of the fluorescence signal derived from the fluorescent probe that binds to the envelope of the virus that separates virus-infected persons from non-virus-infected persons is set as the second threshold value.

[0124] When both a fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below and a fluorescence signal derived from a fluorescent probe that binds to the envelope of the virus are detected, and the value of each signal is equal to or greater than the first threshold value and equal to or greater than the second threshold value, it can be detected or determined that the virus-infected person is highly likely to be infected with the virus.

[0125] In a seventh aspect of the present invention, there is provided a method for detecting viral infection of cells in vitro, the method comprising contacting a compound of formula (1) or a compound of formula (3) with cells suspected of viral infection, and irradiating the cells with light after the contacting, wherein when a fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below is detected, it indicates that the cells are highly likely to be virus-infected.

[0126] Details of each step and the materials used in the seventh aspect of the present invention are as described in the sixth aspect of the present invention.

[0127] In a further embodiment of the seventh aspect of the present invention, when a fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below is detected and the signal value is equal to or greater than a first threshold value, it can be detected or determined that the virus-infected person is highly likely to be infected with the virus.

[0128] In the method for detecting virus particles according to the sixth aspect of the present invention, a fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below and a fluorescence signal derived from a fluorescent probe that binds to the envelope are detected. However, the method for detecting viral infection of cells in vitro according to the present invention is not limited thereto. As another detection method, for example, in the seventh aspect of the present invention, a fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below is detected. According to the seventh aspect of the present invention, it is possible to detect viral infection of cells in vitro using only the fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below.

[0129] In an eighth aspect of the present invention, there is provided a kit for detecting virus particles, particularly coronaviruses, in a sample, the kit comprising a compound of formula (1) below or a compound of formula (3) below, a fluorescent dye that binds to the envelope of the virus, and instructions for detecting virus particles.

[0130] The details of the compound of formula (1) and the compound of formula (3) are as described with respect to the compound of formula (1) of the first aspect and the method of the fourth aspect.

[0131] In a preferred embodiment, the kit comprises a container containing the compound of formula (1) or the compound of the following formula (3). The kit may comprise a pipette, a dropper or other sample handling substrates.

[0132] The kit may comprise a positive control and / or a negative control sample. The positive control sample may contain RNA and / or RNA coexisting with DNA. The negative control sample may be a sample containing DNA without RNA, or a sample containing no nucleic acid at all.

[0133] The kit may comprise one or more additional dyes or stains. For example, the kit may comprise a nucleic acid stain. The kit may comprise a cell-permeable nucleic acid stain for discriminating between live and dead cells.

[0134] The kit may further comprise water, buffer, buffer salts, surfactants, detergents, salts, polysaccharides or other materials commonly used in bioassays. The kit may comprise a solvent such as a water-soluble, water-insoluble, or water-soluble / water-insoluble solvent system.

[0135] In one embodiment, when both the fluorescence signal derived from the compound of formula (1) or the compound of the following formula (3) and the fluorescence signal derived from the fluorescent probe binding to the viral envelope are detected, it can be detected or determined that virus particles are present in the sample.

[0136] In a further embodiment, the average value of the fluorescence signal derived from the compound of formula (1) or the compound of the following formula (3) in a virus-infected individual, the median value of the fluorescence signal derived from the compound of formula (1) or the compound of the following formula (3) in a virus-infected individual, or the value of the fluorescence signal derived from the compound of formula (1) or the compound of the following formula (3) that differentiates virus-infected individuals from non-infected individuals is set as the first threshold value. Also, the average value of the fluorescence signal derived from the fluorescent probe that binds to the envelope of the virus in a virus-infected individual, the median value of the fluorescence signal derived from the fluorescent probe that binds to the envelope of the virus in a virus-infected individual, or the value of the fluorescence signal derived from the fluorescent probe that binds to the envelope of the virus that differentiates virus-infected individuals from non-infected individuals is set as the second threshold value.

[0137] When both the fluorescence signal derived from the compound of formula (1) or the compound of the following formula (3) and the fluorescence signal derived from the fluorescent probe that binds to the envelope of the virus are detected, and the value of each signal is equal to or greater than the first threshold value and equal to or greater than the second threshold value, it can be detected or determined that the virus-infected individual is highly likely to be infected with the virus.

[0138] In a ninth aspect of the present invention, there is provided a kit for detecting virus particles, particularly coronavirus, in a sample, the kit comprising a compound of the following formula (1) or a compound of the following formula (3), and instructions for detecting virus particles.

[0139] Details of the kit and the materials used in the ninth aspect of the present invention are as described in the eighth aspect of the present invention.

[0140] In a further embodiment of the ninth aspect of the present invention, when the fluorescence signal derived from the compound of formula (1) or the compound of the following formula (3) is detected and the value of the signal is equal to or greater than the first threshold value, it can be detected or determined that the virus-infected individual is highly likely to be infected with the virus.

[0141] The kit of the eighth aspect of the present invention described above comprises a compound of formula (1) or a compound of formula (3) below, and a fluorescent probe that binds to the envelope. However, the kit for detecting virus particles in a sample of the present invention is not limited to this. For example, the kit of the ninth aspect of the present invention comprises a compound of formula (1) or a compound of formula (3) below. According to the kit of the ninth aspect of the present invention, it is possible to detect virus particles in a sample even with only the fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below.

[0142] The methods of the fourth to seventh aspects and the kits of the eighth and ninth aspects can be applied to the prevention and prevention of virus infection and the visualization of virus attachment in the medical and research fields. Furthermore, it can also contribute to the treatment of virus infection, the development of virus infection prevention technologies, the removal of virus contamination, and the verification of virus inactivation.

[0143] In addition, the present invention can also adopt the following configuration.

[0144] Item 1. A compound of the following formula (1).

[0145]

Chemical formula

[0146]

Chemical formula

[0147] Item 2. The compound of formula (1) is the compound according to Item 1 which is a compound of formula (1a) or formula (1b).

Chemical formula

[0148] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are each independently hydrogen, a hydroxy group, a thiol group, halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.

[0149] Item 3. The compound of formula (1) is the compound according to Item 1 or 2 which is a compound of formula (2a), formula (2b), or formula (2c).

Chemical formula

[0150] Item 4. A fluorescent dye containing the compound according to any one of Items 1 to 3.

[0151] Item 5. The fluorescent dye according to Item 4 which is an RNA detection agent.

[0152] Item 6. Use of the compound according to any one of Items 1 to 3 for intracellular RNA imaging.

[0153] Item 7. A method for detecting RNA in a sample, comprising: mixing the compound according to any one of Items 1 to 3 with a sample containing RNA to form a mixed sample in which the compound is bound to the RNA in the sample, and irradiating the mixed sample with light to detect the RNA in the mixed sample. A method comprising the above steps.

[0154] The method according to item 7, wherein the sample is a living cell.

[0155] Item 9. A kit for detecting RNA in a sample, comprising: The compound according to any one of items 1 to 3, and Instructions for detecting RNA in a sample, A kit comprising the same.

[0156] Item 10. A method for detecting infection with a virus having an envelope, comprising: Contacting the sample suspected of the presence of virus particles with the compound of formula (1) according to any one of items 1 to 3 or the compound of formula (3) below and a fluorescent dye that binds to the envelope of the virus, and After the contact, performing light irradiation on the sample, A method indicating that there is a high possibility that coronavirus particles are present in the sample when both the fluorescence signal derived from the compound of formula (1) or the compound of formula (3) below and the fluorescence signal derived from the fluorescent probe that binds to the envelope of the virus are detected.

[0157]

Chemical formula

[0158] In formula (3), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen, a hydroxy group, a thiol group, a halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.

[0159] Item 11. A method for detecting virus infection having an envelope, comprising: Contacting the compound of formula (1) according to any one of items 1 to 3 or the compound of the following formula (3) with a sample in which the presence of the virus particles is suspected, and after the contact, performing light irradiation on the sample, A method indicating that there is a high possibility that coronavirus particles are present in the sample when a fluorescence signal derived from the compound of formula (1) or the compound of formula (3) is detected.

[0160]

Chemical formula

[0161] In formula (3), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently hydrogen, a hydroxy group, a thiol group, a halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.

[0162] A method for detecting viral infection of cells in vitro, comprising contacting the compound of formula (1) according to any one of items 1 to 3 or the compound of the following formula (3) and a fluorescent dye that binds to the envelope of the virus with cells in which viral infection is suspected, and after the contact, performing light irradiation on the cells, A method indicating that the cells are highly likely to be virus-infected when both a fluorescence signal derived from the compound of formula (1) or the following formula (3) and a fluorescence signal derived from a fluorescent probe that binds to the envelope of the virus are detected.

[0163]

Chemical formula

[0164] In formula (3), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently hydrogen, a hydroxy group, a thiol group, halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.

[0165] Item 13. A method for detecting viral infection of cells in vitro, comprising: contacting a compound of formula (1) according to any one of Items 1 to 3 or a compound of the following formula (3) with cells suspected of viral infection, and irradiating the cells with light after the contact, wherein when a fluorescence signal derived from the compound of formula (1) or the compound of formula (3) is detected, it indicates that the cells are highly likely to be virus-infected.

[0166]

Chemical formula

[0167] In formula (3), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently hydrogen, a hydroxy group, a thiol group, halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.

[0168] A kit for detecting infection by a virus having an envelope, comprising: a compound of formula (1) according to any one of items 1 to 3 or a compound of the following formula (3); a fluorescent dye that binds to the envelope of the coronavirus; and instructions for detecting viral infection of cells. A kit comprising the above.

[0169]

Chemical formula

[0170] In formula (3), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently hydrogen, a hydroxy group, a thiol group, a halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.

[0171] A kit for detecting infection by a virus having an envelope, comprising: a compound of formula (1) according to any one of items 1 to 3 or a compound of the following formula (3); and instructions for detecting viral infection of cells. A kit comprising the above.

[0172]

Chemical formula

[0173] In formula (3), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen, a hydroxy group, a thiol group, halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.

[0174] The disclosures of all patent applications and documents cited herein are hereby incorporated by reference in their entirety.

[0175] The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto.

Examples

[0176] Example 1 Synthesis of a novel fluorescent dye 1. Synthesis of BIOP (Production of BIOP) The method for producing BIOP, which is a compound of formula (2a), is as described above. (Identification of BIOP) The identification of BIOP was performed using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS).

[0177] · Analysis results by nuclear magnetic resonance Apparatus used: Bruker Avance III 500 Magnetic field strength: 500 MHz, spectral width: 3089 Hz The obtained 1H NMR spectrum 1 H-NMR (500 MHz, DMSO-d 6 ): δ 8.61 (d, J = 7.5 Hz, 1H), 8.54 (d, J = 8.5 Hz, 1H), 8.31 (d, J = 8.0 Hz, 2H), 8.02 - 8.00 (m, 1H), 7.76 (m, 1H), 7.68 (t, J = 7.0 Hz, 2H), 7.52 (t, J = 6.0 Hz, 1H), 6.40 (s, 1H), 4.44 (s, 3H), 3.81 (s, 3H) The above 1H NMR spectrum is predicted from the structural formula of the compound of formula (1). 1 The position of H and the integration ratio are consistent with the measured values.

[0178] ·Analysis results by electrospray ionization mass spectrometry Apparatus used: JMS-T100CS manufactured by JEOL ESI-MS spectrum Calculated value (Calcd ([M + )) of the compound of BIOP: 314.1288, measured value (found ([M + )): 314.1176 From the above nuclear magnetic resonance and electrospray ionization mass spectrometry, the structure of BIOP was identified.

[0179] (Measurement results of the characteristics of BIOP) ·Fluorescence measurement Apparatus used: FP-6500 manufactured by JASCO Corporation For the calculation of the fluorescence quantum yield, an ethanol solution of Rhodamine 6G (fluorescence quantum yield φ fl = 0.95, Photochem. Photobiol., 2002, 75, 327.) was used as a reference. ·Fluorescence microscope measurement Apparatus used: Deltavision Elite microscopy system manufactured by GE Healthcare For the detection of the fluorescence signal of BIOP, a TRITC filter was used, and for the detection of the fluorescence signal of SYTO RNAselect, a FITC filter was used. BIOP is almost non-fluorescent on the carrier, but showed a clear light-up response with the addition of total RNA derived from Escherichia coli (excitation wavelength λex is 539.5 nm, emission wavelength λem is 580 nm). Also, the fluorescence quantum convergence φ fl in the RNA-bound state of BIOP was calculated to be 0.42, and the quantum convergence φ flThe quantum yield φ of compound BIQ of formula (4) obtained by combining benzo[c,d]indole and quinoline is 0.0085, which is nearly 50 times that of JO-PRO-1 obtained by combining oxalopyridine and quinoline with a quantum yield φ of 0.38. Therefore, it was unexpectedly found to have excellent fluorescence response ability.

[0180] 2. Synthesis of BIOP[5,4-c] (Production of [5,4-c]) The production method of compound BIOP[5,4-c] which is the compound of formula (2b) is as described above. (Identification of BIOP[5,4-c]) The identification of BIOP[5,4-c] was carried out using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) in the same manner as BIOP.

[0181] 1H NMR spectrum 1 H-NMR (500 MHz, DMSO-d 6 ) d 9.88 (d, J = 7.3 Hz, 1H), 9.41 (s, 1H), 8.74 (d, J = 6.7 Hz, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.19 (d, J = 6.7 Hz, 1H), 7.95 (t, J = 7.6 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.45 (d, J = 7.3 Hz, 1H), 6.35 (s, 1H), 4.33 (s, 3H), 3.77 (s, 3H) ESI-MS spectrum From the nuclear magnetic resonance and electrospray ionization mass spectrometry with a calculated value of 314.1288 and a measured value of 314.1744 for the BIOP[5,4-c] compound, the structure of BIOP[5,4-c] was identified.

[0182] (Characteristic measurement results of BIOP[5,4-c]) Fluorescence measurement and fluorescence microscope measurement were also carried out for BIOP[5,4-c] under the same apparatus and measurement conditions as BIOP. BIOP[5,4-c] is almost non-fluorescent by itself, but showed a distinct light-up response upon the addition of total RNA derived from Escherichia coli (excitation wavelength λex = 530 nm, emission wavelength λem = 570 nm). Also, the fluorescence quantum yield φ fl was calculated to be 0.52 for the RNA-bound state of BIOP[5,4-c], and this quantum yield φ fl of BIOP[5,4-c] was even higher than that of BIOP.

[0183] 3. Synthesis of BIOP[5,4-c]-nBu (Production of BIOP) The production method of BIOP[5,4-c]-nBu, which is the compound of formula (2c), is as described above. (Identification of BIOP[5,4-c]-nBu) The identification of BIOP[5,4-c]-nBu was carried out using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS), similar to BIOP.

[0184] 1 H-NMR (500 MHz, DMSO-d 6 ) d 9.87 (d, J = 7.3 Hz, 1H), 9.49 (d, J = 3.4 Hz, 1H), 8.83 (d, J = 6.7 Hz, 1H), 8.27 (d, J = 8.2 Hz, 1H), 8.21 (d, J = 6.7 Hz, 1H), 7.95 (t, J = 7.8 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.46 (d, J = 7.0 Hz, 1H), 6.35 (s, 1H), 4.57 (t, J = 7.3 Hz, 2H), 3.78 (s, 3H), 1.91-1.97 (m, 2H), 1.30-1.36 (m, 2H), 0.94 (t, J = 7.5 Hz, 3H) ESI-MS spectrum From the nuclear magnetic resonance and electrospray ionization mass spectrometry of the calculated value of 356.1757 and the measured value of 356.1946 or more of the BIOP[5,4-c]-nBu compound, the structure of BIOP[5,4-c]-nBu was identified.

[0185] (Characteristic measurement results of BIOP[5,4-c]-nBU) Using the same apparatus and measurement conditions as for BIOP, fluorescence measurement and fluorescence microscopy measurement were also performed on BIOP[5,4-c]-nBU. BIOP[5,4-c]-nBU was almost non-fluorescent as a single substance, but showed a clear light-up response with the addition of total RNA derived from Escherichia coli (excitation wavelength λex = 530 nm, emission wavelength λem = 570 nm).

[0186] Example 2 Fluorescent response to biologically-derived nucleic acids (Materials and Methods) As samples, bovine thymus DNA (Sigma-Aldrich) or Escherichia coli total RNA (Thermo Fisher Scientific) was used. The sample and BIOP were mixed, and the fluorescence spectrum in PBS buffer was measured. The BIOP concentration was 1 μM, and the nucleic acid concentrations were 0, 10 μM, 100 μM, 1000 μM (M = mol / L per nucleotide).

[0187] (Results) Figures 2A and 2B show the fluorescence response of the fluorescent dye BIOP to the wavelength for nucleic acids derived from living organisms. As shown in Figures 2A and 2B, when no nucleic acid is present, BIOP shows almost no fluorescence, but with the binding to nucleic acids, it was found that the fluorescence intensity near 580 nm increases significantly. This is thought to be due to the suppression of the free rotation between heterocycles via the monomethine of BIOP by the binding to nucleic acids. The fluorescence response was greater for the response to Escherichia coli total RNA than for the response to bovine thymus DNA. This suggests that BIOP shows an RNA-selective fluorescence response.

[0188] Example 3 Fluorescent responses of various fluorescent dyes to Escherichia coli total RNA and bovine thymus DNA, respectively (Materials and methods) Under the same conditions as in Example 2, the fluorescence responses of BIOP[5,4-c] and BIQ were also examined. The concentration of each fluorescent dye was set to 1 mM, and the concentration of Escherichia coli total RNA was set to 100 mM (10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and 1.0 mM EDTA).

[0189] (Results) Figure 3 shows the fluorescence responses of each fluorescent dye of BIOP[5,4-c], BIOP, and BIQ to Escherichia coli total RNA and bovine thymus DNA. The vertical axis is a measure of RNA selectivity. BIOP[5,4-c] and BIOP showed significantly greater fluorescence intensity upon binding to RNA compared to BIQ. Also, BIOP[5,4-c] and BIOP showed RNA selectivity comparable to that of BIQ.

[0190] Example 4 Fluorescent response to synthetic nucleic acids (Materials and Methods) After annealing chemically synthesized DNA (Japan Genetics Laboratory) or chemically synthesized RNA (GeneDesign), it was mixed with BIOP and measured in PBS buffer. The BIOP concentration was set to 5 μM, and the concentration of each synthetic nucleic acid was set to 20 μM.

[0191] (Results) Figures 4A and 4B show the fluorescence intensity with respect to the synthetic nucleic acids. As shown in Figures 4A and 4B, in both the cases of RNA and DNA, the fluorescence response was greater for double-stranded RNA and DNA than for single-stranded RNA and DNA. This suggests that BIOP is inserted between base pairs (intercalation). In terms of the difference in bases, the responses to single-stranded guanine RNA and DNA were greater than those to single-strands consisting of other bases. This suggests the binding of BIOS to guanine quadruplexes.

[0192] Example 5 Photostability of the fluorescent dye (Materials and Methods) BIOP, BIOP[5,4-c], BIOP[5,4-c]-nBU, or SYTO RNASelect™ (Invitrogen), a known green fluorescent dye, was mixed with E. coli total RNA, and the time-dependent change in fluorescence intensity in PBS buffer was measured. The concentrations of BIOS, BIOP[5,4-c], and BIOP[5,4-c]-nBU were 1 μM, and the concentration of E. coli total RNA was 700 μM. The irradiation time with light was 90 minutes. The excitation wavelength λex of BIOP was 539.5 nm, the emission wavelength λem was 579.5 nm, the excitation wavelength λex of BIOP[5,4-c] and BIOP[5,4-c]-nBU was 530 nm, the emission wavelength λem was 570 nm, the excitation wavelength λex of RNAselect was 490 nm, and the emission wavelength λem was 530 nm.

[0193] (Results) Figure 5 shows the change in luminescence intensity over time for RNAselect and BIOP, BIOP[5,4-c], and BIOP[5,4-c]-nBU, respectively. As shown in Figure 5, the luminescence intensity of RNAselect decreased over time (a 38% decrease), while for BIOP, the decrease in luminescence intensity was only 2% 90 minutes after the start of the test compared to the start of the test, and BIOP had extremely high photostability compared to RNAselect, a conventional nucleic acid-binding fluorescent dye. For BIOP[5,4-c] and BIOP[5,4-c]-nBU as well, BIOP[5,4-c] had extremely high photostability comparable to that of BIOP, and both had extremely high photostability compared to RNAselect.

[0194] Example 6 Detection of RNA in cells (Materials and Methods) 1.8×10 5 MCF-7 cells were incubated in RPMI 1640 medium containing 10% FCS at 37 °C with 5% CO 2 . Subsequently, the medium was replaced with the above medium containing 1 μM BIOP, and 5% CO 2、They were incubated at 37°C for 20 minutes. Then, after washing twice with PBS buffer, the cells were observed using a fluorescence microscope in PBS buffer. The imaging conditions for the cells were an exposure time of 0.5 s and a transmittance of 32%.

[0195] (Results) Figure 6A shows the RNA imaging image of BIOP applied to live MCF-7 cells, and Figure 6B shows the fluorescence intensity on the line in Figure 6A (the intensity along the direction from top to bottom in Figure 6A is shown from left to right in Figure 6B). As shown in Figures 6A and 6B, when BIOP was applied to RNA imaging using live MCF-7 cells, it was confirmed that BIOP could selectively stain nucleosomes rich in RNA even at a low concentration (1.0 μM) and for a short time (20 min).

[0196] Figure 7A shows the differential interference contrast (DIC) images, the RNA imaging images of BIOP, and the merged images of the DIC and BIOP RNA imaging images of MCF-7 cells that were fixed and permeabilized, treated with DNA-degrading enzyme (DNase), treated with RNA-degrading enzyme (RNase), and untreated (control). Figure 7B shows the fluorescence intensity on the line in the RNA imaging image of BIOP (the intensity along the direction from left to right in Figure 7A is shown from left to right in Figure 7B). As shown in Figures 7A and 7B, when MCF-7 cells were fixed and permeabilized and further treated with DNA-degrading enzyme (DNase) or RNA-degrading enzyme (RNase), the fluorescence intensity of nucleosomes decreased significantly in the RNase treatment. On the other hand, no effect on the fluorescence of nucleosomes was observed in the DNase treatment. This suggests that the fluorescence signal of nucleosomes is based on the binding to RNA. In summary, in this study, it was found that BIOP can function as a highly bright fluorescent probe applicable to live-cell RNA imaging. Figure 8A shows imaging images of cells stained with BIOP, imaging images stained with RNAselectB, and those stained simultaneously (co-stained) with both (merge). Figure 8B shows the fluorescence intensity when scanning downward from top to bottom along the line segment in the merge image. In Figures 8A and 8B, when the same cells were stained separately and simultaneously with BIOP and RNAselect, BIOP was selective for nucleosomes, while the entire nucleus faintly glowed with RNAselect.

[0197] Example 7 (Materials and Methods) Living MCF-7 cells were stained with BIOP, BIO BIOP[5,4-c], and BIOP[5,4-c]-nBu under the same conditions as in Example 6. The imaging conditions for the cells were an exposure time of 0.2 s and a transmittance of 10%.

[0198] (Results) As a result of imaging, as shown in Figures 9(A)-(F), it was found that any of the probes could stain nucleosomes rich in RNA. It was found that the fluorescence signal from the nucleosomes was strongest for BIOP[5,4-c]-nBu. Furthermore, as shown in Figure 10, as a result of imaging using six cells, when comparing the fluorescence signals derived from nucleosomes, nuclei, and cytoplasm, it was found that for any of the fluorescence probes, the nucleosomes rich in RNA had a higher fluorescence intensity than the nuclei and cytoplasm, indicating nucleosome selectivity. As a result of comparing between the probes, it was found that the nucleosome selectivity was high in the order of BIOP[5,4-c], BIOP[5,4-c]-nBu, and BIOP.

[0199] Example 8 Detection of virus infection (Fluorescent response of BIOP to virus particles purified from cells infected with human influenza coronavirus) (Materials and Methods) LLC-MK2 cells (rhesus monkey kidney-derived cells) were infected with HCoV-229E (human coronavirus), and the recovered virus particles (log TCID50 / mL = 6) were purified using the ViraTrap Virus Purification Kit. The solution obtained by purification was diluted to prepare a dilution series (1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32-fold) of solutions by the two-fold serial dilution method. 10 μL of the solution at each step was mixed with BIOP, and the samples were adjusted to a total of 200 μL each (the final concentration of BIOP was 1.0 μM). After allowing each sample to stand at room temperature for 10 minutes, the fluorescence intensity was measured using a fluorescence plate reader (Molecular Device SpectraMax Gemini device) (detection conditions: excitation wavelength 544 nm, detection wavelength 590 nm). A sample without virus was prepared as a Negative control under the same conditions except for not containing virus and used as a comparative control.

[0200] (Results) Figure 11 shows the fluorescence intensity when BIOP was applied to cells infected with human coronavirus in each dilution series. Mixing a sample containing cells infected with human coronavirus with BIOP showed a significant fluorescence response, indicating that BIOP can visualize human coronavirus. In addition, BIOP showed a fluorescence response dependent on the concentration of virus particles and independent of the concentration of BIOP. This result reveals that BIOP has the property of showing a fluorescence response by rapidly binding to the RNA inside the cell membrane of human coronavirus because of its RNA selectivity. Therefore, it is suggested that BIOP functions as a reagent for detecting RNA encapsulated in human coronavirus.

Claims

1. A method for detecting infection by a virus having an envelope, comprising: contacting a sample suspected of containing virus particles with a cell membrane-permeable and intracellular RNA-selective fluorescent probe which is a compound of the following formula (1) (excluding the compounds of formula (1a) or formula (1b)) and a fluorescent dye that binds to the envelope of the virus having the envelope; and irradiating the sample with light after the contacting; A method for detecting infection by a virus having an envelope, wherein when both a fluorescent signal derived from the fluorescent probe and a fluorescent signal derived from the fluorescent dye that binds to the envelope of the virus are detected, it indicates a high possibility that virus particles having the envelope are present in the sample. 【Chemical 1】 (In formula (1), ring A is [Chemical 2] and R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 and R 11 are each independently hydrogen, a hydroxy group, a thiol group, halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.) 【Chem.】 (In each of formula (1a) and formula (1b), R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 and R11 are independently hydrogen, hydroxy, group, thiol group, halo, alkyl group, alkenyl group, alkynyl group, alkoxy group, aryl group, or amino group.)

2. The method according to claim 1, wherein the compound of formula (1) is a compound of formula (2a), formula (2b) or formula (2c).

3. The method according to claim 1 or 2, wherein the fluorescent dye that binds to the envelope is a peptidic fluorescent dye that binds to lipid packing defects.

4. The method according to any one of claims 1 to 3, wherein the detection of the fluorescent signal is performed by detecting virus particles having an envelope from the ratio of bright spots with strong fluorescence intensity.

5. A kit for detecting infection by a virus having an envelope, comprising: a cell membrane-permeable and intracellular RNA-selective fluorescent probe which is a compound of the following formula (1); a fluorescent dye that binds to the envelope of the virus having the envelope; A kit comprising the above. 【Chemical Formula 3】 (In formula (1), ring A is 【Chemical Formula 4】 and R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 and R 11 are independently hydrogen, a hydroxy group, a thiol group, halo, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, or an amino group.)

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