CN106147752B - RNA fluorescent probe and preparation method and application thereof - Google Patents
RNA fluorescent probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent probe, a preparation method thereof and application thereof in detecting nucleic acid RNA. The probe has a thiazole orange styrene structure shown as a chemical structural formula (I), and is simple and stable in structure and easy to prepare. The invention also discloses that the probe can be used for specifically detecting RNA, and can rapidly detect the RNA in the solution through a fluorescence spectrophotometer or directly through naked eye observation under the irradiation of a fluorescent lamp; it can also be used to detect, label or display the presence and distribution of RNA in living cells. The fluorescent material has high-efficiency and specific recognition capability on nucleic acid RNA, has the advantages of good cell membrane permeability, low phototoxicity, biotoxicity, photobleaching property and the like, and overcomes the defects of high price, high equipment requirement, relatively complex technical operation and the like of other detection methods.
Description
Technical Field
The invention relates to a fluorescent probe, a preparation method thereof and application thereof in detecting RNA in aqueous solution, gel and cells and imaging nucleolus in the cells.
Background
Small molecule probes are developed probes for a specific target biomolecule or bio-ion, and can specifically interact with a specific target molecule and be detected by a special detection technology. Compared with the common detection technology, the probe technology has the advantages of high sensitivity, strong specificity, rapidness, accuracy and the like, and is suitable for molecular imaging and real-time monitoring.
RNA (ribonucleic acid) has important regulation and control functions in the whole process of growth, development and apoptosis of organisms; in the process of occurrence of many diseases, RNA plays a key role, and for example, the occurrence of malignant tumor is closely related to the abnormal expression of RNA. However, the development and application of RNA analysis and detection technology is slow compared to DNA analysis and detection technology.
The RNA fluorescent probe has the advantage of high-selectivity and specificity recognition of RNA in a complex solution system or in a living cell environment, and has good application prospect in the field of RNA analysis and detection. Currently, only RNAselect available from Invitrogen is commercially available as an RNA fluorescent probe. The fluorescent probe has some defects in practical imaging application, such as the defects of slow response speed with RNA, high phototoxicity, poor light stability and the like, and the application value of the fluorescent probe is influenced by the defects. Therefore, the development of the small-molecule fluorescent probe with excellent performance has strong market value.
Disclosure of Invention
The invention aims to provide a fluorescent probe aiming at the defects of the prior art.
Another object of the present invention is to provide a method for preparing the above probe.
It is still another object of the present invention to provide the use of the above probe for detecting RNA in aqueous solutions, gels and cells.
The invention realizes the aim through the following technical scheme:
the invention provides a fluorescent probe, which has a structural formula as follows:
in the formula X-As the anion, there may be mentioned iodide ion, bromide ion, p-toluenesulfonate ion, trifluoromethanesulfonate ion or the like. Said X-Is an anion after N atom methylation.
The invention also provides a preparation method of the probe, which is represented as follows:
the method comprises the following specific steps: firstly, 4-chloro-2-methylquinoline reacts with a methylating reagent to obtain a compound
Then 2-methylbenzothiazole reacts with a methylating agent to obtain
Then will be
And
reacting at a molar ratio of 1: 1-1: 1.3 at 25-100 deg.C for 2-5 hr in the presence of one or more of water, methanol and ethanol to obtain intermediate
Finally will be
Reacting with 4-methylthiobenzaldehyde according to a molar ratio of 1: 2-1: 3 at a reaction temperature of 130-150 ℃ for 2-5 hours in the presence of n-butanol and 4-methylpiperidine or piperidine as a catalyst to obtain the final probe compound
The invention also provides the application of the probe in detecting RNA in aqueous solution, gel and cells.
The fluorescent probe is applied to marking or displaying the distribution of RNA and nucleolus in living cells.
Experimental results prove that fluorescent images marked by the fluorescent probe have obvious green light distribution in cytoplasm and nucleolus regions, which clearly indicates that the probe can specifically image RNA in cytoplasm and nucleolus in living cells, and the fluorescent probe is further proved after comparative experiments with the traditional nucleolus fluorescent probe or cells subjected to RNA enzyme digestion experiments.
The fluorescent probe provided by the invention is a novel RNA selective recognition fluorescent probe molecule, and compared with a fluorescent probe with similar functions, the fluorescent probe provided by the invention has the characteristics of low biological toxicity, good membrane permeability, strong color development, good counterstain compatibility and strong light stability, is expected to be widely applied as an RNA and nucleolus fluorescent probe, and is expected to be developed into a simple and visual biological detection reagent for RNA and nucleolus related physiological and pathological researches.
Drawings
FIG. 1 shows the fluorescence spectra of probe 6a and six nucleic acids, AT, DA21, LQ1, Oxy28, random and RNA, AT a concentration of 1: 1.
FIG. 2 is a curve fitted to the fluorescence data of four-strand DNA, double-strand DNA and RNA titrated by probe 6 a.
FIG. 3 shows fluorescence spectra of RNA titrated by probe 6a
FIG. 4 shows fluorescence spectra of probes 6a titrated RNA for C and (F-F)0)/F0Fitted curve.
FIG. 5 shows the electrophoresis of the probe 6a and DNA and RNA gels.
FIG. 6 is an image of the cell image of the probe 6a and the dye DAPI counterstained PC3 cell
FIG. 7 shows that probes 6a and E36 and RNA dye stained PC3 cells after DNase and RNase treatment
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples. The fluorescence spectrum experiment proves that the compound 6a has a larger electronic conjugated system and plane, so that after the compound and RNA generate specific accumulation action, the fluorescence spectrum is obviously changed, the fluorescence intensity is increased by hundreds of times, even the compound is observed by naked eyes under the irradiation of a common ultraviolet lamp, and simultaneously, the compound has weaker action with other nucleic acids and no obvious fluorescence signal response, so that the probe has good specific identification effect. Therefore, when the probe is mixed with different nucleic acids, the specific action between the probe and the nucleic acid molecule generates the change of fluorescence spectrum when the nucleic acid is RNA. When the nucleic acid is DNA (G-quadruplex, double-stranded, single-stranded), no significant signal change occurs. The compound 6a is taken as an example to illustrate the application of the fluorescent probe in the invention in the detection of RNA in aqueous solution, gel and cells by a fluorescence method (comprising a fluorescence microscope and a fluorescence gel imager)
The first embodiment is as follows: synthesis of Compound 2
In a 25ml round bottom flask 0.2g (1.1236mmol) of 4-chloro-quinaldine was weighed, 6 times the molar amount of methyl iodide was added about 0.96g, and 1.5ml of sulfolane was added, the mixture was heated to 50 ℃ and reacted for 18 hours, then cooled, added with anhydrous ether and shaken, filtered with suction, the solid was washed with anhydrous ether, dried under vacuum and weighed to give 0.345g of compound 2 in 95.8% yield:1HNMR(400MHz，DMSO)δ8.56(d，J＝8.4Hz，1H)，8.46(d，J＝8.3Hz，1H)，8.22(t，J＝8.1Hz，1H)，8.01(t，J＝7.9Hz，1H)，7.55(s，J＝7.4Hz，1H)，4.20(s，3H)，3.74(s，1H)，2.68(s，3H)。
example two: synthesis of Compound 4
0.25g (1.68mmol) of 2-methyl-benzothiazole was weighed into a 25ml round bottom flask, 6 times molar amount of methyl iodide was added thereto, 5ml of absolute ethanol was added thereto, after reaction at 80 ℃ for 15 hours, the reacted solution was cooled to room temperature, then 5ml of each of absolute ethanol and chloroform was added thereto, and after shaking, suction filtration was performed, and the precipitate was washed with a small amount of ethanol and chloroform, and after vacuum drying, 0.448g of compound 4 was obtained as a white powdery solid with a yield of 91.7%:1H NMR(400MHz，DMSO)δ8.44(d，J＝8.1Hz，1H)，8.30(d，J＝8.4Hz，1H)，7.90(t，J＝7.8Hz，1H)，7.81(t，J＝7.7Hz，1H)，4.20(s，3H)，3.54(s，1H)，3.17(s，3H)。
example three: synthesis of Compound 5
0.50g each of the compounds 2 and 4 was weighed and charged into a round-bottomed flask containing 10ml of methanol, and stirred at room temperature for 6 minutesAfter that, 2ml of a 0.5mol/L aqueous sodium hydrogencarbonate solution was added and the mixture was stirred at room temperature for about 1 hour. Adding 4ml of saturated KI solution into the reacted solution, stirring for about 15 minutes, then carrying out suction filtration, washing with 10ml of water and 4ml of acetone to finally obtain brick red solid, and drying to obtain 0.98g of compound 5 with the yield of 81.7%:1H NMR(400MHz，DMSO)δ8.77(d，J＝8.3Hz，1H)，8.18(d，J＝8.7Hz，1H)，8.02-7.96(m，2H)，7.74(d，J＝8.2Hz，2H)，7.59(t，J＝7.7Hz，1H)，7.39(t，J＝7.5Hz，1H)，7.34(s，1H)，6.85(s，1H)，4.07(s，3H)，3.98(s，3H)，2.87(s，3H)。
example four: synthesis of Compound 6a
0.0715g (0.160mmol) of 5 is weighed into a 25ml round-bottom flask, 0.049g of 4-methylthiobenzaldehyde with 2 times of molar weight, 1.5ml of n-butanol and 5 drops of 4-methylpiperidine are added, the mixture is reacted for 3 hours at 130-135 ℃, cooled and filtered, the solid is washed by n-butanol, dried and weighed to obtain 66mg, and the yield is 71%:1H NMR(400MHz，DMSO)δ8.73(d，J＝7.8Hz，1H)，8.14(d，J＝8.4Hz，1H)，8.06-8.02(m，1H)，8.00-7.95(m，1H)，7.87(d，J＝8.5Hz，2H)，7.76-7.68(m，3H)，7.64(s，1H)，7.61-7.55(m，2H)，7.42-7.35(m，3H)，6.87(s，1H)，4.13(s，3H)，3.97(d，J＝3.7Hz，3H)，2.56(s，3H)。
example five: fluorescence spectrum experiment of fluorescent probe 6a on selectivity of different nucleic acids
The fluorescence intensity of each of the 5mM stock solutions was diluted to a concentration of 5uM, and then different types of nucleic acids were added thereto, and the fluorescence intensity was measured with a fluorescence spectrophotometer (slit width 10nm, scanning speed 200nm/min, excitation wavelength 475nm), and it was found that the fluorescence intensity was the strongest after the binding of the fluorescent probe 6a to RNA.
Nucleic acids for use in selective assays
Example six: fluorescence titration experiment of fluorescent probe 6a on different nucleic acids
A5 mM stock solution of the compound was diluted to a concentration of 5. mu.M, placed in a fluorescence spectrophotometer, and the concentration of different nucleic acids in the solution was gradually increased, and fluorescence intensity measurement was performed. The measurement conditions were: the width of the slit is 10nm, the scanning speed is 200nm/min, and the excitation wavelength is 475 nm.
Nucleic acids for use in fluorescence titration experiments
Example seven: determination of RNA detection Limit by fluorescent Probe 6a
Diluting 5mM compound stock solution to 5 μ M, scanning with fluorescence spectrophotometer (slit width 10nm, scanning speed 200nm/min, excitation wavelength 475nm), slowly adding RNA to saturate, and calculating detection limit
LOD＝K×Sb/m
LOD (binding constant of Compound), m is the slope of the line drawn between concentration C and (F-F0)/F0, SbFor the standard deviation of multiple measurements with instrument blanks, the K value is generally taken as 3 according to the International Union of pure and applied chemistry recommendations, with an LOD of 6 μ g/L measured at 6 a.
Example eight: cell imaging experiment of thiazole orange styrene compound
Cells were seeded in 6-well plates to a cell density of about 2X 103one/mL, then 5% CO at 37 ℃2And culturing for 24 hours in the environment. Then, the cell culture medium in the 6-well plate in the previous step was discarded, washed with pre-cooled 1 XPBS 3 times, then 1.5mL of pre-cooled pure methanol was added and left in the dark at room temperature for 1min, finally, the pure methanol was discarded and washed with pre-cooled 1 XPBS 3 times, 1mL of 5. mu.M compound was added and left for 15 min. The compound solution in the 6-well plate of the above step was discarded, washed 3 times with pre-cooled 1 XPBS, 1mL of 1. mu.M DAPI solution was added to the 6-well plate and left at 37 ℃ for 2min, and then washed 6 times with pre-cooled 1 XPBS, each for 5 min. The staining of the cells was observed under an inverted fluorescence microscope.
Example nine: RNase and DNase cell imaging experiment of thiazole orange styrene compound
Cells are first seededIn 6-well plates, the density of cells was made approximately 2X 103one/mL, then 5% CO at 37 ℃2And culturing for 24 hours in the environment. Then, the cell culture medium in the 6-well plate in the previous step was discarded, washed with pre-cooled 1 XPBS 3 times, then 1.5mL of pre-cooled pure methanol was added and left in the dark at room temperature for 1min, finally, the pure methanol was discarded and washed with pre-cooled 1 XPBS 3 times, 1mL of 5uM compound was added and left for 15 min. The compound solution in the 6-well plate from the previous step was discarded, washed 3 times with pre-cooled 1 XPBS, and 1mL of DNase, DNase-Free RNase solution were added to six-well plates at 37 ℃ with 5% CO2Incubate for 3h, discard the enzyme solution in the 6 well plate, wash 3 times with pre-cooled 1 × PBS, soak 5min each time. The staining of the cells was observed under an inverted fluorescence microscope.
Example ten: nucleic acid gel electrophoresis experiment
Firstly, preparing 5 XTBE electrophoresis buffer solution, 10% M/V ammonium persulfate, 6X sample loading buffer solution and methylene dipropionamide (29: 1) (%, M/V), then installing an electrophoresis device and preparing gel solution, pouring gel, taking out a comb and a partition plate after the gel is cooled, putting the comb and the partition plate into an electrophoresis tank until the buffer solution is submerged by 1-2mm, preparing a DNA sample into 5 mu M (the sample loading buffer solution in the mixed solution is 1X), preparing RNA into 5mg/L, respectively adding 10uL into gel point sample holes, switching on the whole electrophoresis apparatus, running for 1h at 45V, running for 3h at 100V, taking out a gel block, then putting the gel block into a coloring agent and a compound for soaking and dyeing, drying after dyeing, and placing the gel block on the gel electrophoresis apparatus for observation.
Claims (6)
1. A thiazole orange styrene RNA fluorescent probe is characterized in that the chemical structural formula is shown as I:
in the formula X—Is iodide ion, bromide ion, chloride ion, p-toluenesulfonic acid ion or trifluoromethanesulfonic acid ion.
2. A method for preparing the RNA fluorescent probe as claimed in claim 1, which comprises the following steps:
reacting 4-chloro-2-methylquinoline with a methylating agent to obtain a compound
Reacting 2-methylbenzothiazole with a methylating agent to obtain
Then will be
Reacting to obtain
Finally will be
Reacting with 4-methylthiobenzaldehyde to obtain final probe compound
3. Use of the fluorescent probe of claim 1 for the preparation of a product for detecting nucleic acid RNA in an aqueous solution.
4. Use of the fluorescent probe of claim 1 for the preparation of a reagent for detecting RNA products in agarose or polyacrylamide gels.
5. Use of a fluorescent probe according to claim 1 for the preparation of a product for the detection of RNA in a cell.
6. Use of a fluorescent probe according to claim 1 for the preparation of a product for staining RNA particles in eukaryotic cells and imaging nucleoli.
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| CN106188031B (en) * | 2016-06-30 | 2019-11-26 | 广东工业大学 | Thiazole orange styrene derivative and preparation method thereof and preparing the application in drug-resistance bacteria medicine |
| JP7254298B2 (en) * | 2017-08-25 | 2023-04-10 | 国立研究開発法人科学技術振興機構 | organic optical materials |
| CN108558853B (en) * | 2018-01-15 | 2020-09-11 | 广东工业大学 | Compound and preparation method and application thereof |
| CN108165045B (en) * | 2018-01-15 | 2021-03-16 | 广东工业大学 | Nucleic acid dye compound and preparation method and application thereof |
| CN109535114B (en) * | 2018-12-27 | 2020-11-20 | 浙江工业大学 | Fluorescent compound sensitive to NaClO and preparation and application thereof |
| CN112410404B (en) * | 2020-05-27 | 2023-05-19 | 江西省肿瘤医院(江西省癌症中心) | Open type two-photon nucleic acid probe and application thereof in FISH |
| CN111665351B (en) * | 2020-06-20 | 2021-12-03 | 江南大学 | Method for quickly and specifically determining RNA content |
| CN112679474B (en) * | 2020-12-08 | 2022-03-04 | 南方医科大学顺德医院(佛山市顺德区第一人民医院) | Ratio type fluorescent probe and preparation method and application thereof |
| CN115433181B (en) * | 2022-10-12 | 2024-03-08 | 河南省农业科学院农业质量标准与检测技术研究所 | Fluorescent probe based on hemicyanine structure, and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5863753A (en) * | 1994-10-27 | 1999-01-26 | Molecular Probes, Inc. | Chemically reactive unsymmetrical cyanine dyes and their conjugates |
| CN102154003A (en) * | 2011-02-12 | 2011-08-17 | 天津城市建设学院 | Novel carbazole-bridge-based fluorescent cyanine dye probe and preparation method thereof |
| CN103788940A (en) * | 2012-11-02 | 2014-05-14 | 国家纳米科学中心 | Aggregation-induced emission fluorescent molecule as well as preparation method and fluorescent dye composition, and application of aggregation-induced emission fluorescent molecule and fluorescent dye composition in mitochondria dyeing |
| CN104650609A (en) * | 2013-11-22 | 2015-05-27 | 沈阳药科大学 | Thiazol orange derivative, manufacturing method and application of thiazol orange derivative used as double helix nucleic acid fluorescence molecular probe |
-
2015
- 2015-04-24 CN CN201510213542.1A patent/CN106147752B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5863753A (en) * | 1994-10-27 | 1999-01-26 | Molecular Probes, Inc. | Chemically reactive unsymmetrical cyanine dyes and their conjugates |
| CN102154003A (en) * | 2011-02-12 | 2011-08-17 | 天津城市建设学院 | Novel carbazole-bridge-based fluorescent cyanine dye probe and preparation method thereof |
| CN103788940A (en) * | 2012-11-02 | 2014-05-14 | 国家纳米科学中心 | Aggregation-induced emission fluorescent molecule as well as preparation method and fluorescent dye composition, and application of aggregation-induced emission fluorescent molecule and fluorescent dye composition in mitochondria dyeing |
| CN104650609A (en) * | 2013-11-22 | 2015-05-27 | 沈阳药科大学 | Thiazol orange derivative, manufacturing method and application of thiazol orange derivative used as double helix nucleic acid fluorescence molecular probe |
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