CN109913206A - A kind of RNA fluorescence probe and its preparation method and application - Google Patents
A kind of RNA fluorescence probe and its preparation method and application Download PDFInfo
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- CN109913206A CN109913206A CN201910240319.4A CN201910240319A CN109913206A CN 109913206 A CN109913206 A CN 109913206A CN 201910240319 A CN201910240319 A CN 201910240319A CN 109913206 A CN109913206 A CN 109913206A
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Abstract
The present invention provides a kind of fluorescence probe for detecting RNA, chemical structural formulas are as follows:.It can be used for detecting, mark or show the presence and distribution of RNA in cell.Fluorescence probe provided by the invention is that a kind of novel RNA fluorescent probe molecule has the characteristics that low bio-toxicity, membrane permeability are good, have a wide range of applications as RNA fluorescence probe with the ratio of fluorescence probe similar in its function.
Description
Technical field
The present invention relates to a kind of detection RNA there is the fluorescence probe with distribution, belongs to small organic molecule fluorescence probe field.
Background technique
In various living matters in the cell, ribonucleic acid (RNA) is important large biological molecule, synthesizes in protein
It plays an important role in the process, wherein transfer RNA (tRNA) carries and shift activated amino acid；MRNA (mRNA) is synthesis
The template of protein；RRNA (rRNA) is the main place of cell synthetic proteins matter.In addition its catalysis biological react,
It is played an extremely important role in the biological processes such as gene expression regulation.RNA be primarily present in nucleus nucleolar zone and
In cytoplasm, positioning, activity, number, form etc. include important life science information, these information and medical diagnosis and
It treats closely bound up.Therefore, the RNA imaging in kernel and cytoplasm has ten to biochemistry, biological medicine, life science etc.
Divide important meaning.
Current Imaging-PAM, in real-time monitoring living cells the form of biomolecule, in terms of obtain
It is widely applied.The sample that Imaging-PAM is had the low damage of high detection sensitivity, biological sample and can be lived with dynamic analysis
Etc. advantages, the disadvantages of it is expensive to overcome other detection methods, and equipment requirement is high, and technical operation is relative complex, obtain life
The favor of the disciplinary studies persons such as object, life, medicine.It is various that different targets can be imaged in living cells to adapt to existing situation
Fluorescence probe have become research hotspot.
Compared with numerous commercialization probes (such as DNA probe), rna probe is considerably less, because existing nucleic acid probe is general
There is biggish compatibility with double-stranded DNA, and small molecule and the mechanism of action of RNA are unclear, so the research of rna probe is ratio
More difficult.Only having Molecular Probe Company (Molecular Probes Co.) to provide one at present can be used for RNA imaging
Probe " SYTO RNA-Select ", but the chemical structural formula of the probe is not announced externally yet.The shortage of RNA visible probe at present
The exploitation of pathological research and drug is limited.So the RNA fluorescence probe of Development of Novel structure and function is compeled in eyebrow
Eyelash.
Summary of the invention
Aiming at the problem that lacking rna probe at present, the present invention provides a kind of fluorescence probe for detecting RNA, has film penetrating
The advantages that property is good, cytotoxicity is small.
It is a further object of the present invention to provide a kind of synthetic method of above-mentioned fluorescence probe, raw material is easy to get, synthesis step letter
It is single, high income.
Another object of the present invention is to provide a kind of application of above-mentioned fluorescence probe RNA distribution in detection cell.
To achieve the above object, the present invention adopts the following technical scheme that.
A kind of fluorescence probe detecting RNA, its chemical name is 1- methyl-4-((E)-3-((E)-1,3,3- trimethyls
Indoline -2- subunit) propyl- 1- alkene -1- base) pyridine -1- salt compounded of iodine, abbreviation BHI, chemical structural formula is as shown in the formula (I):
Formula (I).
The preparation method of above-mentioned fluorescence probe, comprising the following steps:
(1) 4- picoline heats in ethanol with iodomethane reacts, isolated Isosorbide-5-Nitrae-dimethyl-pyridinium iodide after reaction
(compound 1)；
(2) Isosorbide-5-Nitrae-dimethyl-pyridinium iodide and 1,3,3- trimethyl -2- methylene indoline acetaldehyde are dissolved in ethyl alcohol, in pyrroles
It is stirred at room temperature in the presence of alkane, there is solid precipitation, filter to obtain crude product, product, i.e. RNA fluorescence probe are obtained after purification.
In step (1), in 4- picoline with the molar ratio of iodomethane be 1:1.2.
In step (2), mole of Isosorbide-5-Nitrae-dimethyl-pyridinium iodide and 1,3,3- trimethyl -2- methylene indoline acetaldehyde
Than for 1:1；1,4- dimethyl-pyridinium iodide and the molar ratio of pyrrolidines are 1:1-9.
In step (1), reaction temperature is 90 DEG C, and the reaction time is 24-48 hours.
In step (2), method of purification is to recrystallize in ethyl alcohol.
The synthetic route of above-mentioned fluorescence probe is as follows:
。
A kind of application of above-mentioned fluorescence probe in detection, label or display cell in the presence and distribution of RNA.It can be used
Wavelength is the one-photon excitation of 488 nm, and detection wave band is red spectral band 550-650 nm.
Fluorescence probe working principle of the invention is as follows:
Probe of the invention constructs fluorescence probe using indoles as parent, by double bond bridging pyridiniujm.The conjugation of suitable size
Structure and strong electron-withdrawing group ensure that red emission；The specific structure of the probe assigns it can be with RNA trench region
It is mutually chimeric to identify RNA.
The invention has the following advantages that
Fluorescence probe provided by the invention is a kind of novel RNA fluorescent probe molecule, and the ratio of fluorescence probe similar in its function,
Probe of the present invention has the characteristics that low bio-toxicity, membrane permeability are good, has a wide range of applications as RNA fluorescence probe,
It is expected to the physiology and succinct, the intuitive biological detection reagent of pathological research that exploitation is RNA.
Detailed description of the invention
Fig. 1 is compound 11H H NMR spectroscopy；
Fig. 2 is fluorescence probe BHI's1H H NMR spectroscopy；
Fig. 3 is fluorescence probe BHI's13C H NMR spectroscopy；
Fig. 4 is titration fluorescence spectrum picture of the probe BHI to RNA；
Fig. 5 is probe BHI to fixed cell imaging picture；
Fig. 6 is to fix cell imaging picture after probe BHI handles RNase；
Fig. 7 is the cytotoxicity test results of probe BHI.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be further described, but the present invention is not by the limit of following embodiments
System.
The synthesis of 1 fluorescence probe of embodiment
(1) 1,4- dimethyl-pyridinium iodide synthesis:
8 mL ethyl alcohol are added into round-bottomed flask, the 4- picoline of 1 mL is then added, the iodomethane that 0.755 mL is added is molten
Liquid is heated to 90 DEG C of 24 h of reaction, is after completion of the reaction cooled to room temperature reaction system, there is solid precipitation, filters and uses second
Isosorbide-5-Nitrae-dimethyl-pyridinium iodide (compound 1), yield 92% can be obtained in alcohol washing.Its nuclear magnetic resonance spectroscopy is as shown in Figure 1:1H
NMR (400 MHz, DMSO-d6) δ 8.84 (s, 2H), 7.97 (s, 2H), 4.27 (s, 3H), 2.61 (s,
3H)；
(2) synthesis of rna probe BHI:
Take compound 1(0.6 g, 2 mmol) it is added in round-bottomed flask, the ethyl alcohol of 5 mL is added, 1,3, the 3- front threes of 1 eq are added
Base -2- methylene indoline acetaldehyde is added the pyrrolidines of 1 eq, 12 h is stirred at room temperature, and has a large amount of solids to be precipitated, and filtering can obtain slightly
Product, crude product are reshuffled three times with dehydrated alcohol, are then recrystallized in ethanol, obtain sterling BHI, yield 48%.Its nuclear-magnetism hydrogen
Spectrum and carbon spectrum are as shown in Figures 2 and 3:1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 6.9 Hz, 2H),
8.05 – 7.95 (m, 1H), 7.85 (d, J = 6.8 Hz, 2H), 7.42 (d, J = 7.2 Hz, 1H), 7.25
(d, J = 7.5 Hz, 1H), 7.06 (s, 1H), 7.01 (s, 1H), 6.33 (s, 1H), 5.79 (s, 1H),
4.07 (s, 3H), 3.34 (s, 3H), 1.62 (s, 6H).
13C NMR (101 MHz, DMSO) δ 165.52, 153.56, 144.37, 143.19, 139.77, 128.56,
127.70, 122.04, 120.17, 116.67, 108.63, 97.68, 47.09, 47.09, 47.05, 46.24,
46.23, 46.05, 40.64, 40.43, 40.22, 40.02, 39.81, 39.60, 39.39, 29.95, 28.65。
Response of 2 fluorescence probe of embodiment to RNA
The DMSO mother liquor of the fluorescence probe prepared in configuration case study on implementation 1, concentration are 1 mM.Then 5 μ L probe mother liquors are taken respectively
It is added in 5 mL volumetric flasks, the RNA solution with concentration different volumes is added in each volumetric flask, it is finally fixed with PBS buffer solution
Hold 5 mL, the equivalent proportion of the concentration and concentration and probe concentration that make RNA is respectively 5,20,40,60,100,200,300,400,500,
600.Then fluorescence detection (500 nm of excitation wavelength) is carried out.Using wavelength as abscissa, fluorescence intensity is that ordinate makees Fig. 4.By
Figure is as can be seen that the fluorescence of probe itself is very weak, and when RNA is 600 with probe equivalent proportion, fluorescence intensity enhances 4 times.
Fluorescence imaging of the 3 fluorescence probe BHI of embodiment to cell
The fluorescence probe DMSO mother liquor prepared in embodiment 1 is prepared, concentration is 1 mM.20 μ L are taken to be diluted with l mL culture medium again,
Obtain final concentration of 20 μM of probe dilution liquid.3 are washed with PBS after inoculated cell is handled 30 min with l mL paraformaldehyde
It is secondary, 3 min then are handled with the TritonTMX-100 of 0.5 mL 5%, 30 min are finally incubated at room temperature in probe dilution liquid,
It is washed 3 times with PBS, the cell of adherent growth is placed on glass slide；Then light field imaging and fluorescence imaging are carried out with fluorescence microscope
(excitation wavelength 488 nm, emission band 550-650 nm), as a result as shown in Figure 5: fluorescence probe BHI can dye fixed cell
Cytoplasm and kernel, issue red fluorescence.
It is washed 3 times after inoculated cell is handled 30 min with l mL paraformaldehyde with PBS, then with 0.5 mL's 5%
TritonTMx-100 handles 3 min, and the RNase (5 mg/mL) for adding 3 μ L handles 2 h, finally the room in probe dilution liquid
Temperature is incubated for 30 min, is washed 3 times with PBS, the cell of adherent growth is placed on glass slide；Then with fluorescence microscope carry out light field at
Picture and fluorescence imaging (excitation wavelength 488 nm, emission band 550-650 nm), as a result as shown in Figure 6: compared with Fig. 5, using
After the fixed cell of RNase processing, intracellular red fluorescence obviously dies down.
Toxicity of the 4 probe BHI of embodiment to cell
In the HeLa cell inoculation to the partial hole of 96 orifice plates for being 8000/mL by cell density, remaining hole then uses PBS buffer solution
Filling, according to following conditions in CO2Incubated cell in incubator: experimental group is small with the culture medium incubation 2 of the BHI containing 5 μM
When, 24 hours and the cell sample after 36 hours, control group is the cell sample that dyestuff is not added, and blank group is PBS buffer solution
Sample.After the completion of being incubated for, cell culture fluid is changed with fresh culture medium, and is added 10 μ L's in each culture hole
MTT, then incubated cell 4 hours.After the completion of incubation, culture medium is removed, the DMSO of 200 μ L is added in every hole, and shakes it with shaking table
10 min are to dissolve first a ceremonial jade-ladle, used in libation.Absorbance of each hole at 570 nm, the survival rate (Survival of cell are tested using microplate reader
Rate it) can be calculated by following formula:
Wherein, AsampleFor experimental group absorbance, AcFor control group absorbance, AbFor the absorbance of blank group.When being incubated for probe
Between be abscissa, be that ordinate makees Fig. 7 using cell survival rate: cell survival rate still up to 90% after 36 h of dyeing illustrates probe pair
The toxicity of living cells is very low.
Claims (5)
1. a kind of fluorescence probe for detecting RNA, its chemical name is 1- methyl-4-((E)-3-((E)-1,3,3- trimethyls two
Hydrogen indoles -2- subunit) propyl- 1- alkene -1- base) pyridine -1- salt compounded of iodine, chemical structural formula is as shown in the formula (I):
Formula (I).
2. a kind of preparation method of fluorescence probe as described in claim 1, which comprises the following steps:
(1) 4- picoline heats in ethanol with iodomethane reacts, isolated Isosorbide-5-Nitrae-dimethyl-pyridinium iodide after reaction；
(2) Isosorbide-5-Nitrae-dimethyl-pyridinium iodide and 1,3,3- trimethyl -2- methylene indoline acetaldehyde are dissolved in ethyl alcohol, in pyrroles
It is stirred at room temperature in the presence of alkane, there is solid precipitation, filter to obtain crude product, product, i.e. RNA fluorescence probe are obtained after purification.
3. preparation method according to claim 2, which is characterized in that in step (1), in 4- picoline with iodomethane
Molar ratio is 1:1.2；
In step (2), the molar ratio of Isosorbide-5-Nitrae-dimethyl-pyridinium iodide and 1,3,3- trimethyl -2- methylene indoline acetaldehyde is
1:1；1,4- dimethyl-pyridinium iodide and the molar ratio of pyrrolidines are 1:1-9.
4. preparation method according to claim 2, which is characterized in that in step (1), reaction temperature is 90 DEG C, when reaction
Between be 24-48 hours.
5. a kind of fluorescence probe as described in claim 1 is in detection, label or display cell in the presence and distribution of RNA
Using.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114014848A (en) * | 2021-12-03 | 2022-02-08 | 云南大学 | RNA fluorescent probe and preparation method and application thereof |
| CN115745969A (en) * | 2022-11-24 | 2023-03-07 | 常熟理工学院 | Fluorescent probe, preparation method and application thereof |
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| CN102516792A (en) * | 2011-12-16 | 2012-06-27 | 江南大学 | Structural general formula of cyanine dye for detecting RNA |
| CN103265947A (en) * | 2013-06-03 | 2013-08-28 | 山东大学 | Indolpyridine type fluorescent probe for imaging RNA and nucleolus in living cell |
| CN108997312A (en) * | 2018-08-09 | 2018-12-14 | 济南大学 | A kind of RNA fluorescence probe positioning mitochondria |
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2019
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Patent Citations (4)
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| US3317266A (en) * | 1963-05-16 | 1967-05-02 | Ibm | Electrochromic light valve |
| CN102516792A (en) * | 2011-12-16 | 2012-06-27 | 江南大学 | Structural general formula of cyanine dye for detecting RNA |
| CN103265947A (en) * | 2013-06-03 | 2013-08-28 | 山东大学 | Indolpyridine type fluorescent probe for imaging RNA and nucleolus in living cell |
| CN108997312A (en) * | 2018-08-09 | 2018-12-14 | 济南大学 | A kind of RNA fluorescence probe positioning mitochondria |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114014848A (en) * | 2021-12-03 | 2022-02-08 | 云南大学 | RNA fluorescent probe and preparation method and application thereof |
| CN114014848B (en) * | 2021-12-03 | 2022-04-29 | 云南大学 | RNA fluorescent probe and preparation method and application thereof |
| CN115745969A (en) * | 2022-11-24 | 2023-03-07 | 常熟理工学院 | Fluorescent probe, preparation method and application thereof |
| CN115745969B (en) * | 2022-11-24 | 2023-12-29 | 常熟理工学院 | Fluorescent probe, preparation method and application thereof |
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