CN108949154B - Application of small molecule containing naphthalimide as fluorescent probe in RNA detection and imaging - Google Patents
Application of small molecule containing naphthalimide as fluorescent probe in RNA detection and imaging Download PDFInfo
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- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
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- C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
Abstract
The invention belongs to the technical field of cell imaging, and particularly relates to application of a small molecule containing naphthalimide as a fluorescent probe in RNA detection and imaging. The small molecular compound containing the naphthalimide is combined with RNA to have a fluorescence enhanced response, but the response to DNA under the same condition is very weak, so that the small molecular compound has very good selectivity in a solution state, can be used as a probe for distinguishing the RNA and the DNA, and can eliminate the DNA interference selective recognition RNA in the solution state and living cells; moreover, the small molecular compound can rapidly penetrate through cell membranes and nuclear membranes of living cells within two minutes, realizes fluorescence imaging of RNA in cytoplasm and nucleus, and has good biocompatibility.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to application of a fluorescent probe containing naphthalimide in RNA detection and imaging. The probe can exclude DNA interference from selectively recognizing RNA in a phosphate buffer solution and cells; in living cells, compounds can rapidly cross the cell and nuclear membranes enabling fluorescence imaging of all RNA in the cytoplasm and nucleus.
Background
RNA is a single strand formed by transcription by taking one strand of DNA as a template and taking base complementary pairing as a principle, has the main function of realizing the expression of genetic information on protein, is a bridge for converting the genetic information into expression, and is also involved in the regulation and catalysis of chemical reaction in cells. Changes in the amount of RNA expression and changes in the expression position are important markers for metabolic abnormalities in organisms. Thus, imaging RNA within living cells not only aids in understanding the nature of life activities but also improves the accuracy of disease detection.
At present, probes for imaging cellular endogenous RNA can be mainly classified into four categories: (1) fluorescence-labeled in situ hybridization probes (FISH) are sequences of nucleic acids labeled with fluorophores that are complementary to different portions of the targeting RNA. They are a well-developed imaging technique, but the method has a high background and can only image in fixed cells. (2) Molecular Beacons (MBs) that do not fluoresce when they do not recognize RNA; after binding of RNA, fluorescence is restored. They solve the problem of high background signal of in situ hybridization probes, but the materials are difficult to enter living cells. (3) The nano molecular beacon refers to a molecular beacon loaded with nanoparticles. The nano particles can be used as a quenching agent and a carrier at the same time, so that the synthesis cost can be reduced. (4) Small molecule fluorescent probe, organic small molecule compound. Its advantages are simple synthesis, adjustable fluorescent spectrum and high development potential.
The existing small-molecule RNA fluorescent probe mainly comprises two major types of cyanine dyes and organic metal complexes. In imaging RNA in living cells, two problems are prevalent: (1) the selectivity is not high, especially for DNA that is structurally similar to RNA. It is difficult to find a type of fluorescent probe which can well distinguish the two types of fluorescent probes; (2) the poor permeability of membranes, i.e., the difficulty of these probes in rapid RNA imaging across cell and nuclear membranes, requires long incubation times during imaging, which limits the follow-up of RNA kinetics. Meanwhile, due to the structural characteristics of cyanine dyes, the cyanine dyes have the characteristics of small Stokes shift, easiness in photobleaching and the like. Due to the structural characteristics of the organic metal complex with positive charges, RNA and DNA with negative charges are difficult to distinguish. Therefore, development of other classes of organic small molecule RNA probes is highly desirable.
The naphthalimide compound is a good fluorescent probe because of good light stability and large Stokes shift. However, no RNA response has been reported for the existing naphthalimide compounds. We have discovered a class of RNA fluorescent probes that can respond with increased fluorescence to RNA but weaker to DNA. Meanwhile, the probe can rapidly enter a living cell, and fluorescence imaging of RNA in the cell is realized.
Disclosure of Invention
The invention aims to provide a small molecular compound containing naphthalimide, which is used as a fluorescent probe for RNA detection and imaging.
The invention relates to a small molecular compound containing naphthalimide, which takes naphthalimide as a fluorescent parent, wherein 4-site of the naphthalimide is modified by amino, and nitrogen atoms on the naphthalimide parent are covalently connected with free amino compounds; the structural formula is as follows:
the molecular general formula is: [ R ]1R2N-C12NO2-R3-NH2](ii) a In the formula, R1，R2, R3Is alkyl chain containing 1-8 carbons, halogen substituted alkyl chain or ether oxygen chain.
Experiments show that the small molecular compound containing the naphthalimide is combined with RNA to have fluorescence enhanced response, but the response to DNA under the same conditions is very weak, so that the small molecular compound has good selectivity in a solution state, can be used as a probe for distinguishing RNA and DNA, and can eliminate DNA interference in a solution state and living cells to selectively recognize RNA.
Experiments show that the small molecular compound containing the naphthalimide can rapidly penetrate through cell membranes and nuclear membranes of living cells within two minutes in living cells, realizes fluorescence imaging of RNA in cytoplasm and cell nucleus, and has good biocompatibility.
First, the fluorescence spectrum of the small molecule compound titrated with the saccharomyces baeckensis RNA in phosphate buffer was examined, see fig. 1. The compound is found to have continuous enhancement of fluorescence intensity with the addition of the Becker yeast RNA, and finally the fluorescence intensity is enhanced to 34 times of the original intensity, and meanwhile, the maximum emission wavelength is blue-shifted from 545 nm to 532 nm, which shows that the compound has fluorescence enhancement response to the RNA and the maximum emission wavelength is blue-shifted. FIG. 2 is a fluorescence titration spectrum of the compound with DNA under the same conditions, and FIG. 2 shows that the fluorescence intensity is enhanced with the addition of calf thymus DNA, but is finally increased by about 5 times, and the maximum emission wavelength is not shifted with the addition of DNA. The description combines fig. 1 and fig. 2: the compound has better fluorescence response characteristics to RNA relative to DNA, and the difference in maximum emission wavelength indicates that the compound interacts with DNA and RNA in different binding modes.
Then, a live cell imaging assay was performed, see fig. 3. The test shows that: the compound can penetrate through cell membranes and nuclear pores within 30 seconds to dye nucleolus, nucleolus fluorescence is saturated within 60 seconds, and punctate fluorescence in cytoplasm is gradually enhanced and saturated within 120 seconds as time goes on. This experiment demonstrates that this compound is a probe for rapid imaging.
FIG. 4 shows results of the selectivity test of RNase validation probes in cells. FIG. 4 shows a more significant decrease in the overall fluorescence intensity of cells co-cultured with RNase; whereas the decrease in fluorescence intensity was smaller in the cells co-cultured with DNase. FIG. 4 shows that the commercial probe Syto for RNA live cells has the same tendency as this probe. This result may indicate that the probe is more easily degraded by rnase. Syto is a commercially available RNA probe with high selectivity for RNA in cells. The probes of the invention also have similar selectivity in cells.
FIG. 5 shows the results of cytotoxicity test of the test probe in cervical cancer cells by MTT method. The cell is incubated with the cervical cancer cell for 6 hours in the experimental concentration, and the survival rate of the cell is over 90 percent. This indicates that the probe is substantially non-toxic to cervical cancer cells, confirming the biocompatibility of the probe.
The invention has the advantages that: the small molecule compound has the advantage of RNA specific response, and can distinguish RNA from DNA. The probe can selectively respond to RNA in phosphate buffer, the fluorescence is enhanced to 34 times of the original fluorescence, and the DNA is enhanced by only 5 times under the same condition, so that the RNA and the DNA in the solution can be distinguished. In the living cell imaging research, the probe can rapidly enter the living cell within 2 minutes, and the rapid fluorescence imaging of RNA in the living cell is realized. RNA enzyme degradation experiments verify that the probe has good selectivity in aqueous solution and also has good selectivity in cell imaging. Meanwhile, the probe has higher cell survival rate to HeLa cells, and the biocompatibility of the probe is shown.
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FIG. 1 shows fluorescence titration spectra of probe molecule C against RNA. The excitation wavelength of the fluorescence spectrum was 450 nm, the collection range of emission wavelengths was 470 to 650 nm, the concentration of the probe was 10. mu.M, the test conditions were 20 mM phosphate buffer pH 7.2, and the concentration of RNA was measured as an average molecular weight of 25000 daltons.
FIG. 2 shows the selectivity of probe molecule C for RNA. The fluorescence titration spectra of the probe under the same conditions with DNA are shown. The excitation wavelength of the fluorescence spectrum was 450 nm, the collection range of emission wavelength was 470 to 650 nm, the concentration of probe C was 10. mu.M, the test conditions were 20 mM phosphate buffer pH 7.2, and the concentration of DNA was measured as average molecular weight 25000 daltons.
FIG. 3 is live cell imaging of probe C in HeLa cells. The probe concentration was 5. mu.M, and the probe was dissolved in a DMEM high-glucose medium and co-cultured with HeLa cells at 37 ℃. A 488 nm laser is selected, and the collection wave band is 515-565 nm. The photographs are fluorescence photograph and bright field photograph of the HeLa cells co-cultured with the probe for 0 second, 30 seconds, 60 seconds, 90 seconds and 120 seconds.
FIG. 4 shows RNase degradation experiments. HeLa cells were plated on 6-well plates, incubated with 5. mu.M probe C for 5 minutes, then fixed with ice methanol, and incubated with cells for 4 hours with RNase or DNase, respectively. Fluorescence photographs were taken of the samples without treatment, with DNase treatment, and with RNase treatment. A 488 nm laser is selected, and the collection wave band is 515-565 nm.
FIG. 5 shows the MTT method for testing the cytotoxicity of probe molecule C in cervical cancer cells. Cell viability was verified for 0, 1, 2.5, 5, 7.5, 10, 15, 20 and 30 μ M probe concentrations, respectively, incubated with HeLa cells for 6 hours.
FIG. 6 shows probe molecule D (R)1 = R2 = CH3，R3 = CH2CH2CH2CH2CH2CH2) Fluorescence titration spectra of RNA. The excitation wavelength of the fluorescence spectrum was 450 nm, the collection range of the emission wavelength was 470 to 700 nm, the concentration of the probe was 10. mu.M, the test condition was 20 mM phosphate buffer pH 7.2, and the concentration of RNA was in mass units. mu.g/mL.
FIG. 7 shows the selectivity of probe molecule D for RNA. Fold change in fluorescence for probe D titrated with RNA or DNA under the same conditions is shown. The excitation wavelength of the fluorescence spectrum was 450 nm, the collection range of the emission wavelength was 470 to 650 nm, the concentration of probe D was 10. mu.M, and the test condition was 20 mM phosphate buffer pH 7.2.
FIG. 8 is live cell imaging of Probe D in HeLa cells. The probe concentration was 5. mu.M, and the probe was dissolved in a DMEM high-glucose medium and co-cultured with HeLa cells at 37 ℃. A 488 nm laser is selected, and the collection wave band is 515-565 nm. The photographs are a fluorescence photograph (left) after incubating the probe D and the HeLa cells for 5 minutes, a bright field photograph (center) and a photograph obtained by superimposing fluorescence and bright field (right).
FIG. 9 is a graph showing the cytotoxicity of probe D in cervical cancer cells tested by the MTT method. Cell viability was verified for 0, 1, 2.5, 5, 7.5, 10, 15, 20 and 30 μ M probe concentrations, respectively, incubated with HeLa cells for 6 hours.
FIG. 10 shows a probe molecule E (R)1 = R2 = CH3，R3 = CH2CH2CH2CH2CH2CH2) Fluorescence titration spectra of RNA. The excitation wavelength of the fluorescence spectrum was 450 nm, the collection range of the emission wavelength was 470 to 700 nm, the concentration of the probe was 10. mu.M, the test condition was 20 mM phosphate buffer pH 7.2, and the concentration of RNA was in mass units. mu.g/mL.
FIG. 11 shows the selectivity of probe molecule E for RNA. Fold change in fluorescence of probe E titrated with RNA or DNA under the same conditions is shown. The excitation wavelength of the fluorescence spectrum was 450 nm, the collection range of the emission wavelength was 470 to 650 nm, the concentration of probe E was 10. mu.M, and the test condition was 20 mM phosphate buffer pH 7.2.
FIG. 12 is live cell imaging of probe E in HeLa cells. The concentration of the probe E is 5 mu M, and the probe E and HeLa cells are co-cultured at 37 ℃ after being dissolved by a DMEM high-sugar culture medium. A 488 nm laser is selected, and the collection wave band is 515-565 nm. The photographs are a fluorescence photograph (left) after adding probe E and incubating HeLa cells for 5 minutes, a bright field photograph (center) and a photograph in which fluorescence is superimposed on the bright field (right).
FIG. 13 is a graph showing the cytotoxicity of probe E in cervical cancer cells tested by the MTT method. Cell viability was verified for 0, 1, 2.5, 5, 7.5, 10, 15, 20 and 30 μ M probe concentrations, respectively, incubated with HeLa cells for 6 hours.
Detailed Description
The invention is further illustrated by the following examples.
Example 1: a small molecular compound containing naphthalimide, wherein R1 = R2 = CH3，R3 = CH2CH2And is denoted as probe molecule C. The response to RNA in solution was examined.
The probe molecule C was dissolved in 20 mM phosphate buffer to 10. mu.M, the Becker RNA solution was gradually added dropwise, and the change of the fluorescence spectrum with the addition of RNA was observed. In the experiment, the excitation wavelength of the fluorescence spectrum is 450 nm, and the collection range of the emission wavelength is 470-650 nm.
Example 2: selectivity of Probe molecule C for RNA in solution
The probe molecule C was dissolved in 20 mM phosphate buffer solution to 10. mu.M, and gradually added dropwise with calf thymus DNA solution, and the change of the fluorescence spectrum with the gradually added DNA was observed. When tested, the fluorescence spectrum had an excitation wavelength of 450 nm and a collection range of emission wavelengths of 470 to 650 nm.
Example 3: cellular imaging of Probe molecule C in HeLa Living cells
HeLa cells were seeded in 6-well plates and cultured overnight to allow complete adherence. And (3) dissolving the probe by using a serum-free DMEM high-sugar culture medium to prepare a solution with the final concentration of 5 mu M, adding a 6-hole plate, co-culturing the solution and the HeLa cells at 37 ℃, and observing the dyeing condition of the probe molecule C on the HeLa cells at different times by using a laser confocal microscope. In the experiment, a 488 nm laser is selected. The collected wave band is 515 and 565 nm.
Example 4: RNA enzyme degradation experiment verifies selectivity of probe molecule C in cells
Rnases are proteases that specifically degrade RNA without degrading DNA, whereas dnases only degrade DNA without degrading RNA. In this experiment, HeLa cells were first seeded in 6-well plates and cultured overnight, resulting in complete cell adherence. Then 2 mL of ice methanol was added to the 6-well plate and incubated for 5 minutes to fix the cells. And then adding the probe 5 mu M or Syto 1 mu M into different wells for culture. Confocal laser imaging stained cells of fluorescent probes and Syto. Then, RNase or DNase was diluted to 100. mu.g/ml with serum-free medium, and added to different 6-well plates, respectively, and incubated in a 37-degree incubator for 4 hours. And taking a fluorescence imaging picture after RNA enzyme degradation and DNA enzyme degradation by laser confocal again. The results prove that: our fluorescent probe is similar to the commercial RNA live cell dye Syto. That is, the fluorescence signal in HeLa cells has more obvious fluorescence reduction after being incubated by RNase than after being incubated by DNase. This demonstrates that both probes have better selectivity in cells.
Example 5: cytotoxicity of Probe molecule C in cervical cancer cells
The MTT method was used to test the cytotoxicity of probe molecule C in co-incubation with HeLa cells for 6 hours. Cell viability was tested at probe concentrations of 0, 1, 2.5, 5, 7.5, 10, 15, 20 and 30 μ M, respectively.
Example 6: a small molecular compound containing naphthalimide, wherein R1 = R2 = CH3，R3 = CH2CH2CH2CH2CH2CH2And is designated as probe molecule D. The response to RNA in solution was examined.
The probe molecule D was dissolved in 20 mM phosphate buffer to 10. mu.M, the Becker RNA solution was gradually added dropwise, and the change of the fluorescence spectrum with the addition of RNA was observed. In the experiment, the excitation wavelength of the fluorescence spectrum is 450 nm, and the collection range of the emission wavelength is 470-650 nm.
Example 7: selectivity of Probe molecule D for RNA in solution
The probe molecule D was dissolved in 20 mM phosphate buffer solution to 10. mu.M, and gradually added dropwise with calf thymus DNA solution, and the change of the fluorescence spectrum with the gradually added DNA was observed. When tested, the fluorescence spectrum had an excitation wavelength of 450 nm and a collection range of emission wavelengths of 470 to 650 nm.
Example 8: cellular imaging of Probe molecule D in HeLa Living cells
HeLa cells were seeded in 6-well plates and cultured overnight to allow complete adherence. And (3) dissolving the probe by using a serum-free DMEM high-sugar culture medium to prepare a solution with the final concentration of 5 mu M, adding a 6-hole plate, co-culturing the solution and the HeLa cells at 37 ℃, and observing the dyeing condition of the probe molecule D on the HeLa cells after 5 minutes by using a laser confocal microscope. In the experiment, a 488 nm laser is selected. The collected wave band is 515 and 565 nm.
Example 9: cytotoxicity of Probe molecule D in cervical cancer cells
The cytotoxicity of probe molecule D in co-incubation with HeLa cells was tested for 6 hours by the MTT method. Cell viability was tested at probe concentrations of 0, 1, 2.5, 5, 7.5, 10, 15, 20 and 30 μ M, respectively.
Example 10: a small molecular compound containing naphthalimide, wherein R1 = R2 = CH2CH3，R3 = CH2CH2And is designated as probe molecule E. The response to RNA in solution was examined.
The probe molecule E was dissolved in 20 mM phosphate buffer to 10. mu.M, the Becker RNA solution was gradually added dropwise, and the change of the fluorescence spectrum with the addition of RNA was observed. In the experiment, the excitation wavelength of the fluorescence spectrum is 450 nm, and the collection range of the emission wavelength is 470-650 nm.
Example 11: selectivity of Probe molecule E for RNA in solution
The probe molecule E was dissolved in 20 mM phosphate buffer solution to 10. mu.M, and gradually added dropwise with calf thymus DNA solution, and the change of the fluorescence spectrum with the gradually added DNA was observed. When tested, the fluorescence spectrum had an excitation wavelength of 450 nm and a collection range of emission wavelengths of 470 to 650 nm.
Example 12: cellular imaging of Probe molecule E in HeLa Living cells
HeLa cells were seeded in 6-well plates and cultured overnight to allow complete adherence. And (3) dissolving the probe by using a serum-free DMEM high-sugar culture medium to prepare a solution with the final concentration of 5 mu M, adding a 6-hole plate, co-culturing the solution and the HeLa cells at 37 ℃, and observing the dyeing condition of the probe molecule E to the HeLa cells after 5 minutes by using a laser confocal microscope. In the experiment, a 488 nm laser is selected. The collected wave band is 515 and 565 nm.
Example 13: cytotoxicity of Probe molecule E in cervical cancer cells
The MTT method was used to test the cytotoxicity of probe molecule E in co-incubation with HeLa cells for 6 hours. Cell viability was tested at probe E concentrations of 0, 1, 2.5, 5, 7.5, 10, 15, 20 and 30 μ M, respectively.
The fluorescent probe synthesized in the examples has RNA-specific responsiveness. The probe can selectively respond to RNA in a phosphate buffer. But only a low fluorescence enhancement for DNA under the same conditions. The probe can rapidly penetrate through cell membranes and nuclear pores to stain RNA in cell nucleoli and cytoplasm within 2 minutes, so that rapid fluorescence imaging of RNA in living cells is realized, and an RNase degradation experiment verifies the characteristic that the probe selectively stains RNA in cells. Meanwhile, the fluorescent probe has better biocompatibility.
The above-mentioned embodiments are preferred examples of the present invention, and are not intended to limit the present invention, and any modification, change, alteration or substitution made within the principle of the present invention is within the protection scope of the present invention.
Claims (1)
1. The small molecule containing naphthalimide is used as a fluorescent probe for RNA detection and imaging, the naphthalimide is used as a fluorescent parent, amino modification is carried out on the 4-position of the fluorescent parent, and a nitrogen atom on the naphthalimide parent is covalently connected with a free amino compound; the structural formula is as follows:
in the formula, R1，R2, R3Is alkyl chain containing 1-8 carbons, halogen substituted alkyl chain or ether oxygen chain.
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| CN103113284A (en) * | 2013-01-18 | 2013-05-22 | 大连理工大学 | Half cyanine dye compound, preparation method and application of half cyanine dye compound |
| CN103265947A (en) * | 2013-06-03 | 2013-08-28 | 山东大学 | Indolpyridine type fluorescent probe for imaging RNA and nucleolus in living cell |
| CN103710021A (en) * | 2013-12-12 | 2014-04-09 | 大连理工大学 | Fluorescent dye with nitrobenzimidazole as RNA (ribonucleic acid) recognition group as well as preparation method and application of fluorescent dye |
| CN105777637A (en) * | 2014-12-22 | 2016-07-20 | 中国科学院化学研究所 | Water soluble mitochondrial targeting imaging probe and preparation method thereof |
| CN106634964A (en) * | 2016-11-09 | 2017-05-10 | 大连理工大学 | Application of oxazine compound in preparation of near infrared fluorescence probe |
| WO2017125462A1 (en) * | 2016-01-19 | 2017-07-27 | Universiteit Gent | Methods for obtaining colored or chromic substrates |
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| CN103113284A (en) * | 2013-01-18 | 2013-05-22 | 大连理工大学 | Half cyanine dye compound, preparation method and application of half cyanine dye compound |
| CN103265947A (en) * | 2013-06-03 | 2013-08-28 | 山东大学 | Indolpyridine type fluorescent probe for imaging RNA and nucleolus in living cell |
| CN103710021A (en) * | 2013-12-12 | 2014-04-09 | 大连理工大学 | Fluorescent dye with nitrobenzimidazole as RNA (ribonucleic acid) recognition group as well as preparation method and application of fluorescent dye |
| CN105777637A (en) * | 2014-12-22 | 2016-07-20 | 中国科学院化学研究所 | Water soluble mitochondrial targeting imaging probe and preparation method thereof |
| WO2017125462A1 (en) * | 2016-01-19 | 2017-07-27 | Universiteit Gent | Methods for obtaining colored or chromic substrates |
| CN106634964A (en) * | 2016-11-09 | 2017-05-10 | 大连理工大学 | Application of oxazine compound in preparation of near infrared fluorescence probe |
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