CN107723342B - Telomere probe and preparation method and application thereof - Google Patents
Telomere probe and preparation method and application thereof Download PDFInfo
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- CN107723342B CN107723342B CN201710974752.1A CN201710974752A CN107723342B CN 107723342 B CN107723342 B CN 107723342B CN 201710974752 A CN201710974752 A CN 201710974752A CN 107723342 B CN107723342 B CN 107723342B
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
Abstract
The invention discloses a telomere probe, a preparation method and an application thereof, wherein the telomere probe is formed by coupling single-stranded DNA on the surface of a CdSSe/ZnS quantum dot, the telomere probe can be combined to a cell telomere through an in-situ hybridization technology, and the telomere hybridized with the telomere probe is imaged by using a single-molecule positioning technology, so that the detection of the telomere is realized. The invention realizes the specific marking of telomeres, utilizes a monomolecular positioning microscope to image, has higher resolution, overcomes the defect of the fluorescence microscopy technology limited by the optical diffraction limit, and can realize the further research on the telomere structure.
Description
Technical Field
The invention relates to a DNA probe, a preparation method and application thereof, in particular to a telomere probe, a preparation method and application thereof.
Background
Telomeres are a special structure at the end of chromosomes of eukaryotes, and the sequence of the telomeres is a G-nucleotide-rich tandem repeat sequence. The ends of the chromosomal DNA are enlarged and grainy, covering both ends of each chromatid like a cap, so that each chromosome pair has 2 chromatids and 4 telomeres. The ends formed by the structure and chromosome breaks differ greatly, e.g., they do not fuse with each other, and the cell can replicate only when the chromosome ends are structurally intact. In vertebrates, the telomere sequence is a double-stranded structure composed of TTAGGG repeat sequences and their complements, and one single-stranded 3' overhang is outside, constituting an overhang structure composed of hundreds of bases. The overhang structure and the double chain form a circular structure (T-loop) through folding, and the tail end of the chromosome is protected. In the process of DNA replication, genetic information at one end of the chromosome end cannot be replicated, so in ordinary human cells, the end of telomere can be gradually shortened along with periodic replication, the proliferation capacity of the cells is limited, and when the specific limit is reached, the cells stop dividing and die. Telomeres are not only thought to be involved in cellular senescence, but are also involved in many diseases. Therefore, telomere research is of great significance in the field of cancer treatment.
Currently, there are several common methods for studying telomeres: southern blotting, Hybridization Protection Assay (HPA), quantitative PCR (PCR), single telomere length assay (steal), but these methods all suffer from various problems, such as failure to give detailed telomere information at the cellular level, uneven primer annealing that may result in insufficient telomere length measurement and hybridization between the primers themselves. The Fluorescence In Situ Hybridization (FISH) technology utilizes the specificity of telomeres, directly marks probes on telomere sequences, and combines the fluorescence microscope technology, so that the operation is simple, and the small-size telomere probes increase the infiltration degree of entering cells. In general, the fluorescence microscopy technique used in the FISH technique is limited by the optical diffraction limit, and can only reach 200-300 nm. This resolution prevents one from carefully observing the telomeres.
The optical super-resolution imaging breaks through the limit of diffraction limit, and achieves higher spatial resolution. Technologies currently used for super-resolution imaging mainly include 3 types: stimulated emission depletion (STED) technology based on optical modulation and structured light illumination microscopy (SIM) technology as well as light activated positioning microscopy (SMLM) and random optical reconstruction microscopy (STORM) based on single molecule positioning. Compared with STED and SIM technologies, the imaging resolution of a single-molecule positioning microscope (SMLM) is higher, which enables the single-molecule positioning technology to be widely applied in the biomedical field. Therefore, telomere imaging by using a single molecule localization technology is expected to deeply research the structure of telomeres.
Disclosure of Invention
The purpose of the invention is as follows: the telomere probe is provided for overcoming the problems of complex detection steps and inaccurate detection in telomere detection, and accurate telomere detection is realized. The invention also provides a preparation method and application of the telomere probe.
The technical scheme is as follows:the telomere probe is characterized in that single-stranded DNA is coupled on the surface of a CdSSe/ZnS quantum dot; the single-stranded DNA is coupled with a carboxyl ligand on the surface of the CdSSe/ZnS quantum dot through an amino group at the 5' end; the single-stranded DNA sequence is 3' -AATCCCAATCCCAATCCCTTTTT- (NH)2)6-5’。
The preparation method of the telomere probe comprises the following steps:
(1) phase inversion of oil phase quantum dots: dissolving the oil phase CdSSe/ZnS quantum dots in chloroform, adding mercaptopropionic acid, uniformly mixing, adding tetramethylammonium hydroxide to adjust the pH value of the solution, and stirring for reaction; after the reaction is finished, adding water into the solution, stirring, standing for layering, and taking supernatant; adding absolute ethyl alcohol into the supernatant for precipitation, centrifuging, and removing the supernatant to obtain a precipitate as a water-phase CdSSe/ZnS quantum dot; the oil-phase CdSSe/ZnS quantum dots are converted into the water-phase CdSSe/ZnS quantum dots, so that the CdSSe/ZnS quantum dots meet the requirements of a single-molecule positioning technology on a fluorescent probe, namely the quantum dots have fluorescent scintillation behavior under single-wavelength excitation in an aqueous solution or a phosphate buffer solution.
(2) Synthesis of telomere probe: and (2) dissolving the water-phase CdSSe/ZnS quantum dots obtained in the step (1) in an aqueous solution, adding NHS and EDC solutions, adding DNA single chains, and reacting to obtain the telomere probe.
While the aqueous solution may be replaced with PBS solution.
NHS for N-hydroxy succinimide, EDC for 1- (3-two amino propyl) -3-ethyl carbodiimide hydrochloride, for activating carboxyl.
In the step (1), the mass-to-volume ratio of the oil phase CdSSe/ZnS quantum dot to the mercaptopropionic acid is 25:1-5 mg/mL.
In the step (1), the pH value of the solution is adjusted by using tetramethylammonium hydroxide, so that the pH value of the reaction solution is more than or equal to 11.
In the step (2), the mass molar ratio of the water phase CdSSe/ZnS quantum dots, NHS and EDC is 100-300:1:1 g/mol.
In the step (2), the mass molar ratio of the water-phase CdSSe/ZnS quantum dots to the single-stranded DNA is 50-150:1 g/mol.
When the oil phase quantum dots are subsequently synthesized, pretreatment is needed, the oil phase quantum dots are firstly dissolved in absolute ethyl alcohol, are uniformly shaken and then are centrifugally precipitated, and the obtained precipitate is subjected to subsequent tests.
According to the invention, the oil-phase CdSSe/ZnS quantum dots are converted into the water-phase CdSSe/ZnS quantum dots, and the dosage of reactants can be controlled to realize complete conversion.
The telomere probe is applied to telomere detection. The method comprises the following steps: (1) fixing the cells, adding TritonX-100, and then washing with a PBS solution; (2) adding RNA enzyme into the cells treated in the step (1) for enzymolysis, and then adding pepsin for enzymolysis; (3) dehydrating the cells treated in the step (2) by using ethanol, heating, and adding a telomere probe for hybridization; (4) and (4) carrying out super-resolution microscopic imaging on the cells treated in the step (3) to detect telomeres.
The telomere probe is used for detecting telomeres, and is a telomere super-resolution imaging method based on a fluorescence in situ hybridization technology, and the specific method is as follows:
inoculating cells into an eight-well plate, incubating for 24h, removing the culture solution, adding 4% paraformaldehyde (0.1% glutaraldehyde in 4% paraformaldehyde solution) to fix the cells for 20min, and washing with phosphate buffer solution (PBS, pH 7.4, 10mmol/L) for 2 times; adding 100-; adding 100-200 μ L100 μ g/mL-200 μ g/mL RNase, placing in a 37 ℃ incubator for reaction for 20-30min, and washing with PBS solution for 2 times; adding 100-; performing gradient dehydration with 70% ethanol, 85% ethanol and pure anhydrous ethanol; placing the eight-hole plate in an oven at 85 deg.C, and heating for 5-10min to denature cell DNA; adding 100-150 mu L of 0.1-0.3 mu g/mL telomere probe solution, reacting at 85 ℃ for 10-20min, transferring to a constant temperature box at 37 ℃ for overnight hybridization, and sealing the eight-well plate in the process to prevent the solution from evaporating; discarding the redundant probe solution, placing the eight-well plate in a constant-temperature water bath shaking table at 55-60 ℃, and gently washing for 2 times by using PBS (phosphate buffer solution) containing 0.1% Tween 20, wherein the washing duration time is 10-20min each time; and finally, discarding the cleaning solution, adding a PBS solution or deionized water, and performing super-resolution microscope imaging.
According to the invention, a Fluorescence In Situ Hybridization (FISH) technology is utilized, a CdSSe/ZnS quantum dot is used as a fluorophore for a telomere probe, and the telomere probe is hybridized to a telomere in a cell to realize the fluorescence labeling of the telomere; the single molecule positioning technology is utilized to carry out super-resolution positioning and imaging on the telomere hybridized with the probe, and the spatial resolution of the single molecule positioning technology can reach 50nm, thereby realizing super-resolution imaging which breaks through the optical diffraction limit (200-300 nm). The single molecule positioning super-resolution optical imaging technology is a Single Molecule Localization Microscopy (SMLM) technology.
In the present invention, "%" is a concentration by mass unless otherwise specified.
Has the advantages that: the invention adopts the CdSSe/ZnS quantum dots with fluorescence scintillation effect in the water phase as the fluorescent group of the telomere probe, meets the application requirement of the single molecule positioning technology, has high probe quantum yield, more switching times and good light stability, and improves the resolution of single molecule positioning imaging; the telomere probe disclosed by the invention performs super-resolution imaging on the telomere, the resolution can reach 50nm, and the careful imaging observation of the telomere can be realized; the telomere probe overcomes the defects that in the traditional FISH technology, a fluorophore is an organic dye molecule, is easy to bleach during single-molecule positioning imaging, needs two beams of laser for irradiation and needs a toxic imaging buffer solution.
Drawings
FIG. 1 is a fluorescent scintigram of a telomere probe of the invention;
FIG. 2 is an optical super-resolution image of telomeres detected by the telomere probe of the present invention.
Detailed Description
Firstly, the source of raw materials
1. The PBS buffer solution is the PBS buffer solution with the pH value of 7.4 and the concentration of 10 mmol/L;
2. the oil phase CdSSe/ZnS quantum dots are provided by nano-crystal technology company Limited;
3. DNA strands are available from Biotechnology engineering (Shanghai) Inc.;
4. the tumor cell is SKBR3 cell;
5. the 2 XSSC solution is Saline sodium citrate buffer, which is sourced from Xiamen Kazakh Biotech limited;
6. the remaining materials were obtained commercially.
Preparation of telomere Probe
Example 1: taking 0.5mL of oil phase CdSSe/ZnS quantum dot solution (the concentration of the oil phase CdSSe/ZnS quantum dot solution is 5mg/mL), dissolving in 1.5mL of absolute ethyl alcohol, shaking uniformly, centrifuging at 8000round/min for 20min, and centrifuging once. Removing supernatant, adding 2mL of trichloromethane and 100 mu L of mercaptopropionic acid, uniformly stirring, adding 200 mu L of tetramethylammonium hydroxide, adjusting the pH value of the solution to enable the pH value of the solution to be more than or equal to 11, violently stirring for 1.5-2h, adding 2mL of deionized water, stirring, standing for layering, taking an upper layer, adding absolute ethyl alcohol with the volume being three times that of the reaction solution, centrifuging at the rotating speed of 8000rmp/min for 20min, centrifuging once, washing the obtained precipitate with absolute ethyl alcohol once to obtain water-phase CdSSe/ZnS quantum dots, dispersing the prepared 2.5mg of water-phase CdSSe/ZnS quantum dot precipitate in 2mL of water to obtain a water-phase CdSSe/ZnS quantum dot solution, and storing at 4 ℃ for more than 48h for later use. 25 μ L of the aqueous CdSSe/ZnS quantum dot solution was dissolved in 450 μ L of an aqueous solution, and 15 μ L of a 10mmol/L solution of NHS and 15 μ L of a 10mmol/L solution of EDC were added, and 50 μ L of a 10mmol/L solution of DNA single strand was added, followed by reaction at 4 ℃ overnight. Centrifuging the reactant for 20min at 6000round/min by using a 10KDa ultrafiltration tube, performing ultrafiltration and purification for 3 times, and removing redundant quantum dots to obtain the telomere probe. The fluorescence characteristics of the prepared telomere probe are shown in FIG. 1.
Example 2: taking 0.5mL of oil phase CdSSe/ZnS quantum dot solution (the concentration of the oil phase CdSSe/ZnS quantum dot solution is 5mg/mL), dissolving in 1.5mL of absolute ethyl alcohol, shaking uniformly, centrifuging at 8000round/min for 20min, and centrifuging once. Removing supernatant, adding 2mL of trichloromethane and 500. mu.L of mercaptopropionic acid, uniformly stirring, adding 200. mu.L of tetramethylammonium hydroxide, adjusting the pH value of the solution to enable the pH value of the solution to be more than or equal to 11, violently stirring for 1.5-2h, adding 2mL of deionized water, stirring, standing for layering, taking an upper layer, adding absolute ethyl alcohol with the volume being three times that of the reaction solution, centrifuging at 8000rmp for 20min, centrifuging, washing the obtained precipitate with absolute ethyl alcohol once to obtain an aqueous phase CdSSe/ZnS quantum dot, dispersing the prepared 2.5mg aqueous phase CdSSe/ZnS quantum dot precipitate in 2mL of water to obtain an aqueous phase CdSSe/ZnS quantum dot solution, and storing at 4 ℃ for more than 48h for later use. mu.L of the aqueous CdSSe/ZnS quantum dot solution was dissolved in 450. mu.L of an aqueous solution, and 15. mu.L of a 10mmol/L solution of NHS and 15. mu.L of a 10mmol/L solution of EDC were added, and 30. mu.L of a 10mmol/L solution of DNA single strand was added, followed by reaction at 4 ℃ overnight. Centrifuging the reactant for 20min at 6000round/min by using a 10KDa ultrafiltration tube, performing ultrafiltration and purification for 3 times, and removing redundant quantum dots to obtain the telomere probe. The telomere probe prepared in example 2 has the same properties as those of example 1.
Example 3: taking 0.5mL of oil phase CdSSe/ZnS quantum dot solution (the concentration of the oil phase CdSSe/ZnS quantum dot solution is 5mg/mL), dissolving in 1.5mL of absolute ethyl alcohol, shaking uniformly, centrifuging at 8000round/min for 20min, and centrifuging once. Removing supernatant, adding 2mL of trichloromethane and 300 mu L of mercaptopropionic acid, uniformly stirring, adding 200 mu L of tetramethylammonium hydroxide, adjusting the pH value of the solution to be more than or equal to 11, vigorously stirring for 1.5-2h, adding 2mL of deionized water, stirring, standing for layering, taking an upper layer, adding absolute ethyl alcohol with the volume being three times that of the reaction solution, centrifuging at the rotating speed of 8000round/min for 20min, centrifuging once, washing the obtained precipitate with absolute ethyl alcohol once to obtain water-phase CdSSe/ZnS quantum dots, dispersing the prepared 2.5mg water-phase CdSSe/ZnS quantum dot precipitate in 2mL of water to obtain a water-phase CdSSe/ZnS quantum dot solution, and storing at 4 ℃ for more than 48h for later use. mu.L of the aqueous CdSSe/ZnS quantum dot solution was dissolved in 450. mu.L of an aqueous solution, and 15. mu.L of a 10mmol/L solution of NHS and 15. mu.L of a 10mmol/L solution of EDC were added, and 32. mu.L of a 10mmol/L solution of DNA single strand was added, followed by reaction at 4 ℃ overnight. Centrifuging the reactant for 20min at 6000round/min by using a 10KDa ultrafiltration tube, performing ultrafiltration and purification for 3 times, and removing redundant quantum dots to obtain the telomere probe. The telomere probe prepared in example 3 has the same properties as those of example 1.
Third, fluorescent in situ hybridization reaction
1. Preparation of aqueous telomere Probe solution
The telomere probe sample prepared in example 1 was dissolved in PBS solution, and the concentration of the telomere probe was 0.2. mu. mol/L.
2. Adding 4% paraformaldehyde into an eight-hole plate with cells to fix the cells for 20min, and washing with PBS for 2 times; adding 150 μ L of 1% TritonX-100, slightly shaking the cells for 30min to increase the permeability of the cells, and washing with PBS solution for 2 times; adding 150 μ L RNase solution 100 μ g/ml, placing in 37 deg.C incubator for reaction for 20min, and washing with PBS solution for 2 times; adding 45 deg.C preheated 150 μ L0.005% pepsin solution (solvent is 10mmol HCl) and placing in 37 deg.C incubator for reaction for 5min, and washing with PBS solution for 2 times; then dehydrating with 70% ethanol solution, 85% ethanol solution and 100% anhydrous ethanol respectively; then placing the eight-hole plate in an oven at 85 ℃ for 5min to denature the DNA; finally, 150. mu.L of 0.2. mu. mol/L telomere probe was added thereto, reacted at 85 ℃ for 10min, and then transferred to a 37 ℃ incubator for hybridization overnight.
The reactant is washed by a prepared washing solution (2 times SSC solution contains 0.1 percent of Tween-20) at 55-60 ℃ for 2 times by slight shaking, each time is 10min, then the washing solution is added into the reactant at room temperature and stands for 10min, and then 100 and 200 mu L of PBS solution are added for imaging of the super-resolution microscope. The results are shown in FIG. 2.
It can be clearly seen from fig. 1 and its partially enlarged diagram that the fluorescence intensity of the telomere probe significantly fluctuates with time (i.e., fluorescence blinking phenomenon), which indicates that the prepared telomere probe is suitable for single molecule positioning imaging. As can be seen from FIG. 2, the telomere probe prepared by the invention can be imaged under the resolution of 50nm, the intensity is stable, and the spatial distribution of telomeres can be clearly observed on an SMLM image.
Claims (1)
1. A telomere probe is characterized in that the telomere probe is single-stranded DNA coupled on the surface of a CdSSe/ZnS quantum dot; the single-stranded DNA is coupled with a carboxyl ligand on the surface of the CdSSe/ZnS quantum dot through an amino group at the 5' end; the single-stranded DNA sequence is 3' -AATCCCAATCCCAATCCCTTTTT- (NH)2)6-5’；
The telomere probe is prepared by the following steps:
(1) dissolving the oil phase CdSSe/ZnS quantum dots in chloroform, adding mercaptopropionic acid, adjusting the pH value of the solution by using tetramethylammonium hydroxide, and stirring for reaction; after the reaction is finished, adding water into the solution, stirring, standing for layering, and taking supernatant; adding absolute ethyl alcohol into the supernatant, precipitating, centrifuging, and removing the supernatant to obtain a precipitate as a water-phase CdSSe/ZnS quantum dot; the mass-to-volume ratio of the oil phase CdSSe/ZnS quantum dots to the mercaptopropionic acid is 25:1-5 mg/mL;
(2) dissolving the water-phase CdSSe/ZnS quantum dots obtained in the step (1) in an aqueous solution, adding NHS and EDC, stirring, adding a DNA single strand, and reacting to obtain a telomere probe; the mass molar ratio of the water phase CdSSe/ZnS quantum dots, NHS to EDC is 100-300:1:1 g/mol/mol; the mass molar ratio of the water-phase CdSSe/ZnS quantum dots to the single-stranded DNA is 50-150:1 g/mol.
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| CN108642139A (en) * | 2018-05-21 | 2018-10-12 | 东南大学 | A method of detecting telomerase activation using super-resolution imaging |
| CN110333371A (en) * | 2019-06-21 | 2019-10-15 | 武汉科技大学 | A kind of gold nano-probe preparation facilities and preparation method thereof |
| CN112592963A (en) * | 2021-01-04 | 2021-04-02 | 东南大学 | Telomere and centromere super-resolution imaging method and probe thereof |
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| CN101381600A (en) * | 2008-10-22 | 2009-03-11 | 中国科学院上海技术物理研究所 | Preparation method of biocompatible aqueous phase quantum point |
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| CN101381600A (en) * | 2008-10-22 | 2009-03-11 | 中国科学院上海技术物理研究所 | Preparation method of biocompatible aqueous phase quantum point |
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