CN112111267A - AIE probe for nucleic acid identification and preparation method and application thereof - Google Patents

AIE probe for nucleic acid identification and preparation method and application thereof Download PDF

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CN112111267A
CN112111267A CN202010830904.2A CN202010830904A CN112111267A CN 112111267 A CN112111267 A CN 112111267A CN 202010830904 A CN202010830904 A CN 202010830904A CN 112111267 A CN112111267 A CN 112111267A
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熊海
肖秋芸
赵烜
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Abstract

The invention discloses an AIE probe for nucleic acid identification and a preparation method and application thereof. The structural general formula of the AIE probe is shown as one of formulas (I), (II) and (III):
Figure DDA0002637930250000011
in formulas (I), (II) and (III), R is independently one of A, T, C, G, U. The AIE probe is a novel fluorescent probe for selectively identifying ribonucleic acid, can be combined with cell nucleus after penetrating cells, emits blue fluorescence due to the aggregation-induced emission principle, and has the advantages of chemical stability, non-quenching and good biocompatibility. Can be widely used for nucleic acid detection,Cellular imaging, and other related biological assays.

Description

AIE probe for nucleic acid identification and preparation method and application thereof
Technical Field
The invention relates to the technical field of fluorescent probes, in particular to an AIE probe for nucleic acid identification and a preparation method and application thereof.
Background
Nucleic acids, including ribonucleic acids and deoxyribonucleic acids, are important biological macromolecules in cells and play important roles in storing genetic information and regulating life activities. The detection and localization of nucleic acids is important in biological diagnostics. Conventional nucleic acid dyes, such as ethidium bromide, can achieve nucleic acid staining by intercalating into DNA double-stranded structures or single-stranded RNA with higher order structures. Ethidium bromide is the most widely used nucleic acid dye with strong binding capacity to nucleic acid and stable fluorescence signal, but the potential carcinogenicity caused by the change of the DNA structure is still controversial. Similar problems exist with other fluorescent dyes used for cellular imaging, such as DAPI (4, 6-diamidine-2-phenylindole), and DAPI dyes are easily quenched, requiring the addition of special anti-quenchers in conventional staining.
Probes designed with aggregation-induced emission (AIE) materials are more favorable for biological imaging than aggregation-quenched (ACQ) fluorescent probes due to their special optical properties, i.e., a sharp increase in fluorescence in the aggregated state. Such as Tetraphenylethylene (TPE), is a typical AIE molecule consisting of a center of carbon-carbon double bonds with four peripheral benzene rings. When its motion is blocked, fluorescence is significantly enhanced. Therefore, when the TPE is combined with the target substance to generate fluorescence, the concentration of the target substance can be preliminarily judged according to the fluorescence intensity. Many studies have demonstrated that TPE and its derivatives are non-toxic to cells and therefore can be widely used in biological assays. So far, no report has been made on nucleoside-AIE fluorescent probes having direct nuclear localization effect and in vitro nucleic acid detection capability.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide an AIE probe for nucleic acid identification, a preparation method and an application thereof, and aims to solve the problems that the existing nucleic acid probe has toxicity to cells and is quenched in fluorescence in an aggregation state.
The technical scheme of the invention is as follows:
in a first aspect of the present invention, an AIE probe for nucleic acid recognition is provided, wherein the structural general formula of the AIE probe is represented by one of formulas (I), (II) and (III):
Figure BDA0002637930230000021
in formulas (I), (II) and (III), R is independently one of A, T, C, G, U.
Alternatively, in formulas (I), (II), and (III), R is independently one of C, U.
In a second aspect of the present invention, there is provided a method for preparing the nucleic acid-recognizing AIE probe of the present invention, comprising the steps of:
under the catalysis of palladium (phosphine), the 5-iodine-deoxynucleoside and 4-ethynyl-tetraphenylethylene are subjected to coupling reaction to obtain a product.
Alternatively, the method for preparing the nucleic acid-recognized AIE probe comprises the steps of: under the catalysis of palladium (phosphine), performing coupling reaction on 5-iodine-2-deoxycytidine and 4-ethynyl-tetraphenylethylene to obtain 2' -deoxy-5-triphenylvinylbenzene-o-ethynyl cytosine;
the above chemical reaction formula is shown as follows:
Figure BDA0002637930230000022
optionally, the preparation method of the nucleic acid-recognized AIE probe specifically comprises the steps of: mixing 5-iodine-2-deoxycytidine, 4-ethynyl-tetraphenylethylene, palladium tetratriphenylphosphine and cuprous iodide, adding anhydrous DMF (dimethyl formamide) for dissolving, then adding triethylamine, and carrying out coupling reaction under stirring to obtain the 2' -deoxy-5-triphenylvinylbenzene-o-ethynyl cytosine.
Alternatively, the method for preparing the nucleic acid-recognized AIE probe comprises the steps of: under the catalysis of palladium (phosphine), performing coupling reaction on 5-iodine-2-deoxyuracil and 4-ethynyl-tetraphenylethylene to obtain 2' -deoxy-5-triphenylvinylbenzene-o-ethynyluracil;
the above chemical reaction formula is shown as follows:
Figure BDA0002637930230000031
optionally, the preparation method of the nucleic acid-recognized AIE probe specifically comprises the steps of: mixing 5-iodine-2-deoxyuracil, 4-ethynyl-tetraphenylethylene, palladium tetratriphenylphosphine and cuprous iodide, adding anhydrous DMF (dimethyl formamide) for dissolving, then adding triethylamine, and carrying out coupling reaction under stirring to obtain 2' -deoxy-5-triphenylvinylbenzene-o-ethynyl uracil.
In a third aspect of the invention, there is provided a use of the AIE probe of the invention in nucleic acid detection.
In a fourth aspect of the invention, there is provided the use of an AIE probe of the invention for nuclear localization.
In a fifth aspect of the invention, there is provided a use of the nucleic acid-recognized AIE probe of the invention in cell imaging.
Has the advantages that: the AIE probe is a novel fluorescent probe for selectively identifying ribonucleic acid, can be combined with cell nucleus after penetrating cells, emits blue fluorescence due to the aggregation-induced emission principle, and has the advantages of chemical stability, non-quenching and good biocompatibility. Can be widely used for nucleic acid detection, cell imaging and other related biological detection.
Drawings
In FIG. 1, A is a chemical reaction formula of 2 '-deoxy-5-triphenylvinylbenzene-o-ethynylcytosine (dC-TPE), and B is a chemical reaction formula of 2' -deoxy-5-triphenylvinylbenzene-o-ethynyluracil (dU-TPE).
FIG. 2 shows dC-TPE probes1H-NMR spectrum.
FIG. 3 shows dU-TPE probes1H-NMR spectrum.
FIG. 4 is a graph of fluorescence spectra of ctDNA at different concentrations and a non-linear fit of the concentrations to fluorescence values for the systems of dC-TPE and dU-TPE at 5. mu.M concentration.
FIG. 5 is a PAGE gel electrophoresis of protoplasmic gene DNA of human histone H3 at different concentrations of 0, 10, 25, 50, 100ng after staining dC-TPE and dU-TPE at 10. mu.M concentration.
FIG. 6 is a graph showing fluorescence spectra of different concentrations of ribonucleic acid in yeast and a non-linear fit of the concentrations to fluorescence values for the 5. mu.M concentration of dC-TPE and dU-TPE systems.
FIG. 7 is a laser confocal diagram of L929 cells after staining with dC-TPE and dU-TPE at a concentration of 5. mu.M (emission wavelength Em:470 nm; excitation wavelength Ex:375 nm).
FIG. 8 is a graph showing the effect of varying concentrations of dC-TPE and dU-TPE on cell viability.
Detailed Description
The present invention provides an AIE probe for nucleic acid identification, and a preparation method and an application thereof, and the present invention will be described in further detail below in order to make the objects, technical solutions, and effects of the present invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides an AIE probe for nucleic acid recognition, which not only can be specifically combined with DNA or RNA in vitro, but also can smoothly pass through a cell membrane to act on living cells and be positioned in a cell nucleus area. The emission spectrum has a maximum at 470nm under 375nm excitation light. The probe has no obvious toxicity to living cells, and is a living cell probe with practical application potential. Wherein the structural general formula of the AIE probe is shown as one of formulas (I), (II) and (III):
Figure BDA0002637930230000051
in formulas (I), (II) and (III), R is independently A, T, C, G, U, namely R is independently any base.
The AIE probe provided by the embodiment of the invention is a novel fluorescent probe for selectively identifying ribonucleic acid, can be combined with a cell nucleus after penetrating through a cell, emits blue fluorescence due to a gathering induced luminescence principle, and has the advantages of chemical stability, non-quenching and good biocompatibility. Has the application potential of being widely used for nucleic acid detection, cell imaging and other related biological detection.
In one embodiment, in formulas (I), (II), and (III), R is independently one of C, U.
In a second aspect of the present invention, there is provided a method for preparing the nucleic acid-recognizing AIE probe of the present invention, comprising the steps of:
under the catalysis of palladium (phosphine), the 5-iodine-deoxynucleoside and 4-ethynyl-tetraphenylethylene are subjected to coupling reaction to obtain a product.
Alternatively, the method for preparing the nucleic acid-recognized AIE probe comprises the steps of: under the catalysis of palladium (phosphine), performing coupling reaction on 5-iodine-2-deoxycytidine and 4-ethynyl-tetraphenylethylene to obtain 2' -deoxy-5-triphenylvinylbenzene-o-ethynyl cytosine;
the above chemical reaction formula is shown as follows:
Figure BDA0002637930230000061
optionally, the preparation method of the nucleic acid-recognized AIE probe specifically comprises the steps of: mixing 5-iodine-2-deoxycytidine, 4-ethynyl-tetraphenylethylene, palladium tetratriphenylphosphine and cuprous iodide, adding anhydrous DMF (dimethyl formamide) for dissolving, then adding triethylamine, and carrying out coupling reaction under stirring to obtain the 2' -deoxy-5-triphenylvinylbenzene-o-ethynyl cytosine.
Alternatively, the method for preparing the nucleic acid-recognized AIE probe comprises the steps of: under the catalysis of palladium (phosphine), performing coupling reaction on 5-iodine-2-deoxyuracil and 4-ethynyl-tetraphenylethylene to obtain 2' -deoxy-5-triphenylvinylbenzene-o-ethynyluracil;
the above chemical reaction formula is shown as follows:
Figure BDA0002637930230000062
optionally, the preparation method of the nucleic acid-recognized AIE probe specifically comprises the steps of: mixing 5-iodine-2-deoxyuracil, 4-ethynyl-tetraphenylethylene, palladium tetratriphenylphosphine and cuprous iodide, adding anhydrous DMF (dimethyl formamide) for dissolving, then adding triethylamine, and carrying out coupling reaction under stirring to obtain 2' -deoxy-5-triphenylvinylbenzene-o-ethynyl uracil.
The embodiment of the invention provides an application of the AIE probe in nucleic acid detection. The nucleic acid staining experiment carried out in vitro shows that the AIE probe has staining effect on DNA and RNA, and the fluorescence intensity is enhanced along with the increase of the nucleic acid concentration in a system; the results of PAGE gel staining of DNA and RNA also demonstrate this conclusion.
The embodiment of the invention provides an application of the AIE probe in positioning of cell nucleus. When the AIE probe provided by the embodiment of the invention is applied to cell staining, a laser confocal image after marking shows that the AIE probe is mainly positioned in a cell nucleus and a small amount of the AIE probe is positioned in cytoplasm.
The embodiment of the invention provides an application of the AIE probe for nucleic acid recognition in cell imaging. The AIE probe provided by the embodiment of the invention has better nucleic acid identification property, can be directly used for live cell imaging, and has a stable fluorescence signal. The fluorescence signal increases with the increase of the nucleic acid concentration, and can be used for nucleic acid diagnosis. The AIE probe adopts a natural ribonucleic acid skeleton, and can be further used for other functional modifications to obtain the AIE fluorescent probe with specific functionality.
The invention is further illustrated by the following specific examples.
Example 1
Preparation of 2' -deoxy-5-triphenylvinylbenzene-o-ethynylcytosine (dC-TPE) Probe
In a 100mL two-necked flask, 211mg (0.6mmol,1 equiv.) of 5-iodo-2-deoxycytidine, 320mg (0.9mmol,1.5 equiv.) of 4-ethynyl-tetraphenylethylene, 69.3mg (0.06mmol,0.1 equiv.) of palladium tetratriphenylphosphine and 22.8mg (0.12mmol,0.2 equiv.) of cuprous iodide were dissolved by adding 10mL of anhydrous DMF, and then 0.3mL of triethylamine was added and the reaction was stirred at room temperature for 12 hours. After the reaction is finished, the reaction system is concentrated and dried, and a target product is obtained through column chromatography. FIG. 1 (a) shows the chemical reaction formula of the target product, and FIG. 2 shows the target product1H NMR chart.
Example 2
Preparation of 2' -deoxy-5-triphenylvinylbenzene-o-ethynyluracil (dU-TPE) Probe
Taking 5-iodo-2-deoxyurine218mg (0.6mmol,1 equivalent) of pyrimidine, 320mg (0.9mmol,1.5 equivalents) of 4-ethynyl-tetraphenylethylene, 69.3mg (0.06mmol,0.1 equivalent) of tetrakistriphenylphosphine palladium and 22.8mg (0.12mmol,0.2 equivalent) of cuprous iodide were dissolved in a 100mL two-necked flask, 10mL of anhydrous DMF was added, 0.3mL of triethylamine was then added, and the reaction was stirred at room temperature for 12 hours. After the reaction is finished, the reaction system is concentrated and dried, and a target product is obtained through column chromatography. FIG. 1 (b) shows the chemical reaction formula of the target product, and FIG. 3 shows the target product1H NMR chart.
Example 3
Application of dC-TPE probe and dU-TPE probe in ctDNA detection
Weighing a certain amount of ctDNA freeze-dried powder, dissolving in PBS (pH7.0), setting 12 concentrations in the interval of 0-48 ng/. mu.L, and setting the concentration interval gradient to be 4 ng/. mu.L. 100-fold concentrations of master solutions of dC-TPE and dU-TPE probes (dissolved in DMSO) were added to the above DNA system, and the concentration was adjusted to 5. mu.M. The excitation wavelength was set at 375nm, the relative fluorescence values were scanned over the interval 375nm-640nm, and the correlation between fluorescence intensity values and concentrations at the maximum emission wavelength was fitted. FIG. 4 is a graph of fluorescence spectra and concentration-luminosity fit curves of ctDNA at different concentrations in 5 μ M dC-TPE and dU-TPE systems. In FIG. 4, A and B are fluorescence spectrum scan of 0-48 ng/. mu.L ctDNA under 375nm excitation light and non-linear fitting graph of concentration to fluorescence value under dC-TPE system with 5. mu.M concentration. In FIG. 4, C and D are fluorescence spectra scans of 0-48 ng/. mu.L ctDNA under 375nm excitation light and non-linear fits of concentration to fluorescence value under dU-TPE system with 5. mu.M concentration.
Example 4
Application of dC-TPE and dU-TPE probes in PAGE gel (polyacrylamide gel) detection of DNA
The application method is the same as EB nucleic acid dye. Preparing dC-TPE and dU-TPE probe mother liquor (dissolved in DMSO and 1mM) with the concentration of 100 times, soaking PAGE gel after electrophoresis is completed in TBE buffer solution added with the probe, dyeing for 40 minutes on a shaking table, and directly placing the PAGE gel into a gel imager for imaging analysis. FIG. 5A is a PAGE gel of human histone H3 protogenic DNA at different concentrations of 0, 10, 25, 50, 100ng after 10. mu.M dC-TPE staining. FIG. 5B is a PAGE gel of dU-TPE staining at 10. mu.M concentration followed by human histone H3 protogenic DNA at various concentrations of 0, 10, 25, 50, 100 ng.
Example 5
Application of dC-TPE (dC-TPE) and dU-TPE (dU-TPE) probes in RNA (ribonucleic acid) detection
Weighing a certain amount of yeast ribonucleic acid lyophilized powder, dissolving in PBS (pH7.0), setting 12 concentrations in the interval of 0-400 μ g/μ L, and setting the concentration interval gradient to 20 μ g/μ L. Add 100-fold concentration of dC-TPE and dU-TPE probe stock to the above RNA system, adjusting the probe concentration to 5. mu.M. The excitation wavelength was set at 375nm, the relative fluorescence values were scanned over the interval 375nm-640nm, and the correlation between fluorescence intensity values and concentrations at the maximum emission wavelength was fitted. FIG. 6 is a graph of fluorescence spectra and concentration-luminosity fit curves of RNA at different concentrations in the 5 μ M concentration of dC-TPE and dU-TPE systems. In FIG. 6, A and B are fluorescence spectrum scan of 0-200 μ g/μ L of ribonucleic acid in yeast under excitation light of 375nm and non-linear fit graph of fluorescence value corresponding to concentration under dC-TPE system with concentration of 5 μ M. In FIG. 6, C and D are fluorescence spectra scans of 0-400. mu.g/. mu.L yeast ribonucleic acid under 375nm excitation light and non-linear fit graphs of concentration versus fluorescence value under 5. mu.M dU-TPE system.
Example 6
Application of dC-TPE and dU-TPE probes in living cell imaging
Adding PBS (pH7.0) into DMSO solution containing dC-TPE and dU-TPE probes to prepare solution with concentration of 5 μ M, fixing cultured L929 cells with paraformaldehyde, rinsing for three times, adding PBS solution containing probes for dyeing, and standing overnight to ensure dyeing effect. Generally, the dyeing effect can be very good after 4 hours of dyeing. The staining solution is removed, and the dried slices are directly used for fluorescence microscope and laser confocal observation (emission wavelength Em:470 nm; excitation wavelength Ex:375nm), so that the cell nucleus staining effect is obvious. FIG. 7 is a laser confocal picture of L929 cells after staining with 5mM dC-TPE and dU-TPE (Em:470 nm; Ex:375 nm). A, B, C in FIG. 7 are the bright field image of the L929 cell stained with dC-TPE, the fluorescence imaging effect under 375nm excitation light, and the combination effect of the fluorescence image and the bright field image, respectively, and it can be seen that the nuclear region of the L929 cell has obvious blue excitation light, indicating that dC-TPE has better nuclear localization and imaging effects. D, E, F in FIG. 7 is a bright field image of L929 cells stained with dU-TPE, a fluorescence imaging effect under excitation light of 375nm, and a combination effect of the fluorescence image and the bright field image, respectively, it can be seen that the nuclear region of the L929 cells has more obvious blue excitation light, indicating that dU-TPE has better nuclear localization and imaging effects, but the nuclear localization and staining effects of dU-TPE are inferior to those of dC-TPE under the same concentration. MTT test detection shows that the synthesized dC-TPE and dU-TPE probes have no obvious cytotoxicity to L929 and HEK cells. FIG. 8 shows the effect of MTT on L929 cell viability of dC-TPE and dU-TPE at different concentrations, and the cell viability was still above 90% when the concentration of both probes reached a higher concentration of 20. mu.M, whereas normally only 5-10. mu.M was required for viable cell treatment, and the cell viability was greater than 95%, indicating that neither probe had a significant toxic effect on the cells.
In summary, the invention provides an AIE probe for nucleic acid identification, and a preparation method and application thereof. The AIE probe is a novel fluorescent probe for selectively identifying ribonucleic acid, can be combined with a cell nucleus after penetrating through a cell, emits blue fluorescence due to a gathering-induced luminescence principle, and has the advantages of chemical stability, non-quenching and good biocompatibility. Can be widely used for nucleic acid detection, cell imaging and other related biological detection.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.

Claims (10)

1. An AIE probe for nucleic acid recognition, which is characterized in that the structural general formula of the AIE probe is shown as one of formulas (I), (II) and (III):
Figure FDA0002637930220000011
in formulas (I), (II) and (III), R is independently one of A, T, C, G, U.
2. The nucleic acid-recognized AIE probe according to claim 1, wherein in formulae (I), (II) and (III), R is independently C, U.
3. A method for preparing the nucleic acid-recognized AIE probe according to any one of claims 1 to 2, comprising the steps of:
under the catalysis of palladium (phosphine), the 5-iodine-deoxynucleoside and 4-ethynyl-tetraphenylethylene are subjected to coupling reaction to obtain a product.
4. The method for preparing an AIE probe for nucleic acid recognition according to claim 3, comprising the steps of: under the catalysis of palladium (phosphine), performing coupling reaction on 5-iodine-2-deoxycytidine and 4-ethynyl-tetraphenylethylene to obtain 2' -deoxy-5-triphenylvinylbenzene-o-ethynyl cytosine;
the above chemical reaction formula is shown as follows:
Figure FDA0002637930220000021
5. the method for preparing an AIE probe for nucleic acid identification according to claim 4, comprising the steps of: mixing 5-iodine-2-deoxycytidine, 4-ethynyl-tetraphenylethylene, palladium tetratriphenylphosphine and cuprous iodide, adding anhydrous DMF (dimethyl formamide) for dissolving, then adding triethylamine, and carrying out coupling reaction under stirring to obtain the 2' -deoxy-5-triphenylvinylbenzene-o-ethynyl cytosine.
6. The method for preparing an AIE probe for nucleic acid recognition according to claim 3, comprising the steps of: under the catalysis of palladium (phosphine), performing coupling reaction on 5-iodine-2-deoxyuracil and 4-ethynyl-tetraphenylethylene to obtain 2' -deoxy-5-triphenylvinylbenzene-o-ethynyluracil;
the above chemical reaction formula is shown as follows:
Figure FDA0002637930220000022
7. the method for preparing an AIE probe for nucleic acid identification according to claim 6, comprising the steps of: mixing 5-iodine-2-deoxyuracil, 4-ethynyl-tetraphenylethylene, palladium tetratriphenylphosphine and cuprous iodide, adding anhydrous DMF (dimethyl formamide) for dissolving, then adding triethylamine, and carrying out coupling reaction under stirring to obtain 2' -deoxy-5-triphenylvinylbenzene-o-ethynyl uracil.
8. Use of the AIE probe of any one of claims 1-2 in nucleic acid detection.
9. Use of an AIE probe according to any one of claims 1 to 2 for nuclear localisation.
10. Use of the nucleic acid-recognized AIE probe of any one of claims 1-2 for cellular imaging.
CN202010830904.2A 2020-08-18 2020-08-18 AIE probe for nucleic acid identification and preparation method and application thereof Active CN112111267B (en)

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Publication number Priority date Publication date Assignee Title
CN113789324A (en) * 2021-08-17 2021-12-14 广东省大湾区华南理工大学聚集诱导发光高等研究院 AIE probe, preparation method thereof and application thereof in fluorescent quantitative PCR (polymerase chain reaction) method
CN113956314A (en) * 2021-11-22 2022-01-21 青岛科技大学 Solvent-free nucleic acid flexible fluorescent material and preparation method thereof

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HYUN YI CHO ET AL.: "Synthesis and photophysical study of 2"-deoxyuridines labeled with fluorene derivatives", 《MOLECULES》 *
YOUNG JUN SEO ET AL.: "Quencher-Free, End-Stacking Oligonucleotides for Probing Single-Base Mismatches in DNA", 《ORGANIC LETTERS》 *
赵烜 等: "P-31：聚集诱导发光脱氧尿苷类似物凝胶材料的制备及应用", 《第十一届全国化学生物学学术会论文摘要（第二卷）》 *

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Publication number Priority date Publication date Assignee Title
CN113789324A (en) * 2021-08-17 2021-12-14 广东省大湾区华南理工大学聚集诱导发光高等研究院 AIE probe, preparation method thereof and application thereof in fluorescent quantitative PCR (polymerase chain reaction) method
CN113789324B (en) * 2021-08-17 2023-08-25 广东省大湾区华南理工大学聚集诱导发光高等研究院 AIE probe, preparation method thereof and application thereof in fluorescent quantitative PCR (polymerase chain reaction) method
CN113956314A (en) * 2021-11-22 2022-01-21 青岛科技大学 Solvent-free nucleic acid flexible fluorescent material and preparation method thereof

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