CN112592963A - Telomere and centromere super-resolution imaging method and probe thereof - Google Patents
Telomere and centromere super-resolution imaging method and probe thereof Download PDFInfo
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- CN112592963A CN112592963A CN202110006799.5A CN202110006799A CN112592963A CN 112592963 A CN112592963 A CN 112592963A CN 202110006799 A CN202110006799 A CN 202110006799A CN 112592963 A CN112592963 A CN 112592963A
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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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The invention discloses a telomere and centromere super-resolution imaging method and a probe thereof, wherein the telomere DNA probe comprises 3 repeating units in the sequence, the sequence of the repeating units is shown as SEQ ID NO.1, the 5' ends of the telomere DNA probe and the centromere DNA probe are provided with fluorescent molecules, by means of the reversible combination of the probe DNA sequence, the super-resolution single-molecule positioning imaging of the telomere and the centromere is realized by utilizing total internal reflection illumination to realize the on and off of fluorescent signals of the two probes through the steps of cell fixation, RNA digestion, cell dehydration, hybridization, cleaning and imaging, and the advantages of low experimental cost, short period, good specificity and accuracy are realized.
Description
Technical Field
The invention relates to DNA hybridization and optical super-resolution imaging, and mainly relates to a telomere and centromere super-resolution imaging method and a probe thereof.
Background
Telomeres are a small segment of DNA-protein complex located at the tail end of a linear chromosome of a eukaryotic cell, and are special structures formed by a short multiple non-transcribed sequence (TTAGGG) and a plurality of binding proteins, so that the tail end of the chromosome DNA is protected, and the degradation, the tail end fusion, the abnormal recombination and the like of the chromosome DNA are avoided. In mammals, the existence of telomere can maintain the chromosome end to be stable, and the length of the telomere is continuously shortened along with the increase of the cell division times, so that the DNA and genetic information in the cells can be stably and completely existed. However, with the continuous shortening of telomeres, the genome in the cells cannot be maintained in a stable state, and finally, the cells are aged, dead or cancerized. Therefore, measuring telomere imaging helps to provide important information related to the disease.
The existing telomere and centromere imaging methods mainly comprise: electron microscope imaging, fluorescence staining in combination with optical microscope imaging, and the like. The resolution of the electron microscope can reach 0.1nm, and the details of the sample can be displayed with extremely high resolution. However, electron microscopes also have various problems, mainly that the sample preparation process of electron microscopes is very complicated and harsh, and electron microscopes can only see the structures inside cells, and other technologies are needed for the structure judgment to assist in distinguishing. In the past, researchers have also imaged telomeres using light microscopy, which allows for the labeling of specific cells and even molecules by various fluorescent staining methods. However, this method can only image telomeres and centromeres with a medium resolution of several hundreds of nanometers, and cannot image at a single molecule level, due to the limitation of diffraction or "spreading" of light waves through a small aperture or focusing to a minute spot by the resolution of an optical microscope.
These methods have important contribution in telomere research, but they all have disadvantages, such as low sensitivity, complicated procedure, high cost, insufficient resolution, etc., so that telomere and centromere imaging technologies with high sensitivity, high accuracy and wide applicability need to be designed.
The super-resolution fluorescence microscopic imaging (SMLM) based on single molecule positioning comprises light-activated positioning imaging (PALM) and random optical reconstruction super-resolution imaging (STORM), an organic fluorescent probe and a super-resolution optical microscopic imaging technology are tightly combined together, and the organic fluorescent probe provides a powerful tool for researching the super-resolution imaging.
SMLM imaging can also be performed using complementarity of DNA strands to produce an effect similar to fluorescence molecule blinking. By adjusting the binding strength and concentration of the DNA strands, transient binding can be repeatedly generated, and a fluorescence blinking phenomenon similar to that of a fluorescent dye in STORM can be obtained. And reconstructing the obtained data to obtain a super-resolution image.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the problems of complex operation, low sensitivity and high cost of the existing telomere measurement super-resolution imaging method, the invention provides a telomere and centromere super-resolution imaging method and a probe thereof, and the super-resolution single molecule localized imaging (SMLM) of the telomere and the centromere is realized by means of the reversible combination of DNA sequences.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that: a telomere and centromere super-resolution imaging method, it includes the following steps, (1) add fixative to fix the cell in the cell culture plate inoculated with cell to be measured, carry on the cell membrane to penetrate; (2) adding an RNA inhibitor for RNA digestion; (3) adding pepsin to decompose protein; (4) adding ethanol to dehydrate the cells in a gradient manner; (5) preparing a telomere DNA probe, a centromere DNA probe and a hybridization buffer solution into a probe solution, wherein the sequence of the telomere DNA probe comprises 3 repeating units, the sequence of the repeating units is shown as SEQ ID NO.1, and fluorescent molecules are arranged at the 5' ends of the telomere DNA probe and the centromere DNA probe; (6) adding the probe solution into an eight-hole plate for hybridization; (7) and collecting telomere and centromere fluorescence signals on the cells by using a total internal reflection illumination mode, and collecting fluorescence images and performing super-resolution single molecule positioning imaging.
Preferably, the fluorescent molecule comprises one or more of Cy3 and Cy 5.
Preferably, the sequence of the telomere DNA probe is shown in SEQ ID NO.2, and the sequence of the centromere DNA probe is shown in SEQ ID NO. 3.
Preferably, step (1), the fixing agent comprises 4% paraformaldehyde, and the fixing time is more than 20 min; the cell membrane penetration is carried out by penetrating the membrane with 0.2% triton (TritonX-100) for more than 5 min; in the step (2), the RNA inhibitor is RNaseA solution prepared from PBS, the concentration is 100 mug/mL, and the reaction time is more than 20 min; in the step (3), the pepsin is a pepsin solution with the concentration of 0.005% (w/v), diluted hydrochloric acid with the concentration of 10mM is used for preparation, the diluted hydrochloric acid is heated to 37 ℃ before preparation, the pepsin solution is preheated to 45 ℃ before use, the decomposition temperature is 37 ℃, and the decomposition time is more than 5 min; in the step (4), the gradient dehydration is performed by using 70 percent, 85 percent and 100 percent (v/v) of glacial ethanol in sequence; in the step (5), the formula of the hybridization buffer solution is as follows: 20mM Tris-HCl, pH7.4, 60% (v/v) formamide, 0.1. mu.g/mL salmon sperm DNA; in the step (6), the eight-hole plate is heated to 85 ℃ for 10min, then the DNA telomere probe and the DNA centromere probe are added, and then the hybridization reaction is carried out under the condition of keeping out of the sun, wherein the hybridization temperature is 37 ℃ and the hybridization time is 3 hours.
Preferably, in the step (7), the collecting of telomere and centromere fluorescence signals on the cells utilizes the excitation light with the wavelength of 647nm and 561nm respectively, collects the fluorescence signals with the wavelength of more than 561nm and more than 642nm, and the collecting of the fluorescence image is carried out, wherein the image exposure time is 50 milliseconds, and at least 3000 frames are collected.
As another aspect of the present invention, the present invention provides a probe for telomere and centromere super-resolution imaging, characterized in that: the 5' end of the probe is provided with fluorescent molecules, and the fluorescent molecules comprise one or more of Cy3 and Cy 5; the probes comprise a telomere DNA probe and a centromere DNA probe, the sequence of the telomere DNA probe comprises 3 repeating units, and the sequence of the repeating units is shown as SEQ ID NO. 1.
Preferably, the sequence of the telomere DNA probe is shown in SEQ ID NO.2, and the sequence of the centromere DNA probe is shown in SEQ ID NO. 3.
Compared with the prior art, the telomere and centromere super-resolution imaging method and the probe thereof provided by the invention have the following advantages:
1. the telomere and centromere DNA probes used in the method can be combined with the telomere and the centromere reversibly;
2. the DNA probe used in the invention realizes the on-off of two probe fluorescence signals by utilizing total internal reflection illumination in the SMLM super-resolution imaging process, and does not need special instruments or operation to carry out optical switching;
3. the invention realizes telomere super-resolution imaging by adopting DNA probe in-situ hybridization, and has the advantages of low experiment cost, short period, good specificity and accuracy.
Drawings
FIG. 1 is a schematic diagram of a super-resolution method for imaging telomeres and centromere according to the present invention;
FIG. 2 is a schematic diagram of the structure of telomeres and centromere DNA probes used in the present invention;
FIG. 3 is an image of SMLM super-resolution imaging of telomere and centromere probes under 647nm and 561nm excitation light channels, wherein the left side is a single-molecule localization image of telomere DNA probes, the average intensity of fluorescence is 2.57, the right side is a single-molecule localization image of centromere DNA probes, the average intensity of fluorescence is 3.09, and the scale bar is 1 μm.
Detailed Description
The present invention will be further described with reference to the accompanying drawings.
The raw materials in the invention are as follows:
1. the PBS buffer was pH7.4, PBS buffer at a concentration of 10 mM;
2. the telomere DNAT probe with 5 '-Cy 5 TTTTTCCCTAACCCTAACCCTAA-3' and the centromere DNA probe with the sequence of 5 '-Cy 3 TTTTTAGCTTCTGTCTAGTTT-3' are synthesized by the company Biotechnology engineering (Shanghai);
3. salmon sperm DNA was purchased from Sigma;
4. the remaining materials were obtained commercially.
Example 1:
to the eight-chamber Nunc inoculated with Hela cellsTMLab-TekTMII Chamber SlideTM(hereinafter referred to as an eight-well plate) was added with 200. mu.L of 0.2% (v/v) Triton solution (TX-100), and the mixture was taken out after 1 minute of membrane permeation and washed with PBS buffer; add 200. mu.L of 4% paraformaldehyde (v)V) fixing the fixing solution for 20min, taking out, and washing with PBS buffer solution; 200. mu.L of 0.2% (v/v) triton solution (TX-100) was added, and the membrane was permeabilized for 5 minutes, followed by washing with PBS buffer.
Add 200. mu.L NaBH to eight well plates4Shaking the solution (2mg/mL) at room temperature for 5min, taking out, and washing with PBS buffer solution; adding 200 μ L RNase A solution (100 μ g/mL), reacting at 37 deg.C for 20min, taking out, and washing with PBS buffer solution; adding 200 μ L of 0.005% (w/v) pepsin solution, preheating pepsin solution to 45 deg.C before use, reacting at 37 deg.C for 5min, taking out, and washing with PBS buffer solution; adding 70%, 85% and 100% (v/v) ethanol solution, performing gradient dehydration for 2min each time, and blow-drying the eight-hole plate.
Adding 200. mu.L salmon sperm DNA solution (100. mu.g/mL) into the eight-well plate, reacting at 37 ℃ for 10min, taking out, and washing with PBS buffer; place the eight well plate on a hot plate, preheat at 85 ℃ for 10min, then add 100. mu.L each of the two prepared probe solutions and heat at 85 ℃ for 10 min. And (4) taking out the eight-hole plate after heating, and carrying out light-proof hybridization reaction in a room temperature environment for 3 hours.
After the preparation of the DNA probe in-situ hybridization sample is completed, the eight-hole plate is fixed on a carrying platform of an ultrahigh microscope for super-resolution imaging based on single-molecule positioning. Collecting telomere (Cy5) and centromere (Cy3) fluorescence signals on cells by using a wide-field microscope in a total internal reflection illumination mode by using a 100-fold oil lens, wherein the wavelengths of exciting light are 647nm and 561nm respectively, the excitation intensity is 3%, collecting the fluorescence signals of which the wavelengths are more than 561nm and more than 642nm, collecting the fluorescence signals of which the wavelengths are more than 642nm (Cy5) and more than 561nm (Cy3), and carrying out fluorescence image collection and super-resolution single molecule localization imaging, wherein the image exposure time is 50 milliseconds, and at least 3000 frames are collected.
The telomere DNA probe used in the application is a DNA sequence (SEQ ID NO. 2: 5 '-Cy 5 TTTTTCCCTAACCCTAACCCTAA-3') capable of hybridizing with a telomere, the 5 'end is modified with a fluorescent molecule Cy5, the DNA sequence contains 3 CCCTAA repeating units (SEQ ID NO.1) in total, the process of hybridizing with the telomere is ensured to be reversible, the centromere DNA probe is a DNA sequence (SEQ ID NO. 3: 5' -Cy3TTTTTAGCTTCTGTCTAGTTT-3 ') capable of hybridizing with a centromere, and the 5' end is modified with a fluorescent molecule Cy 3.
The experiment of the invention is carried out through a series of exploration processes, the initial detection effect of the invention is poor, the scintillation phenomenon of a DNA probe can not be observed in a cell nucleus area, and the experimental steps of fixing and membrane penetration are continuously adjusted, wherein the membrane penetration time and the concentration of a TritonX-100 solution are more critical. Through multiple experimental comparisons, when the time for penetrating the membrane is set to be 10min, the concentration of triton (TritonX-100) is set to be 0.2%, in the imaging process, the conditions that the scintillation phenomenon of the DNA probe is disordered, the background noise is strong, and the DNA probe is gathered at the nuclear membrane instead of the cell nucleus occur, and the concentration of the surface DNA probe solution has obvious influence on the imaging. The condition that the concentration of the DNA probe is too high, the background noise is strong, and a large amount of the probe is gathered in a nuclear membrane; if the concentration of the DNA probe is too low, it is possible that some telomeres and centromere sites are not bound by the DNA probe. Finally, after attempting imaging experiments at various concentrations of DNA probe, the concentration of DNA probe selected was 10 nM.
The invention discloses a telomere and centromere super-resolution imaging method, which needs two DNA probes, wherein one DNA probe is a telomere DNA probe capable of hybridizing with a telomere, and the other DNA probe is a centromere DNA probe capable of hybridizing with a centromere. Adding the telomere DNA probe and the centromere DNA probe to the cell sample fixed by the fixing agent, so that the two probes can be respectively hybridized to the telomere and the centromere. The combination of the used telomere DNA probe and the used centromere DNA probe with the telomere and the centromere is reversible, the on and off of fluorescence signals of the two probes are realized by utilizing total internal reflection illumination, and the super-resolution single molecule positioning imaging (SMLM) of the telomere and the centromere is carried out. The method has the advantages of low experiment cost, short period, good specificity and high accuracy.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of southeast
<120> telomere and centromere super-resolution imaging method and probe thereof
<141> 2020-12-24
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Claims (7)
1. A telomere and centromere super-resolution imaging method is characterized in that: comprises the following steps of (a) carrying out,
(1) adding a fixing agent into the cell culture plate inoculated with the cells to be detected to fix the cells, and performing cell membrane penetration;
(2) adding an RNA inhibitor for RNA digestion;
(3) adding pepsin to decompose protein;
(4) adding ethanol to dehydrate the cells in a gradient manner;
(5) preparing a telomere DNA probe, a centromere DNA probe and a hybridization buffer solution into a probe solution, wherein the sequence of the telomere DNA probe comprises 3 repeating units, the sequence of the repeating units is shown as SEQ ID NO.1, and fluorescent molecules are arranged at the 5' ends of the telomere DNA probe and the centromere DNA probe;
(6) adding the probe solution into an eight-hole plate for hybridization;
(7) and collecting telomere and centromere fluorescence signals on the cells by using a total internal reflection illumination mode, and collecting fluorescence images and performing super-resolution single molecule positioning imaging.
2. The method of telomere and centromere super-resolution imaging of claim 1, wherein: the fluorescent molecule comprises one or more of Cy3 and Cy 5.
3. The method of telomere and centromere super-resolution imaging of claim 2, wherein: the sequence of the telomere DNA probe is shown as SEQ ID NO.2, and the sequence of the centromere DNA probe is shown as SEQ ID NO. 3.
4. The telomere and centromere super-resolution imaging method of any one of claims 1 to 3, wherein: step (1), the fixing agent comprises 4% paraformaldehyde, and the fixing time is more than 20 min; the cell membrane penetration is carried out by penetrating the membrane with 0.2% triton (TritonX-100) for more than 5 min; in the step (2), the RNA inhibitor is RNaseA solution prepared from PBS, the concentration is 100 mug/mL, and the reaction time is more than 20 min; in the step (3), the pepsin is a pepsin solution with the concentration of 0.005% (w/v), diluted hydrochloric acid with the concentration of 10mM is used for preparation, the diluted hydrochloric acid is heated to 37 ℃ before preparation, the pepsin solution is preheated to 45 ℃ before use, the decomposition temperature is 37 ℃, and the decomposition time is more than 5 min; in the step (4), the gradient dehydration is performed by using 70 percent, 85 percent and 100 percent (v/v) of glacial ethanol in sequence; in the step (5), the formula of the hybridization buffer solution is as follows: 20mM Tris-HCl, pH7.4, 60% (v/v) formamide, 0.1. mu.g/mL salmon sperm DNA; in the step (6), the hybridization is carried out under the condition of keeping out of the sun, the hybridization temperature is 37 ℃, the hybridization time is 3 hours,
5. the telomere and centromere super-resolution imaging method of any one of claims 1 to 3, wherein: in the step (8), the fluorescence signals of telomere and centromere on the cell are collected, the wavelengths of the excitation light are 647nm and 561nm respectively, the fluorescence signals of more than 561nm and more than 642nm are collected, the fluorescence image is collected, the image exposure time is 50 milliseconds, and at least 3000 frames are collected.
6. A probe for super-resolution imaging of telomeres and centromeres, comprising: the 5' end of the probe is provided with fluorescent molecules, and the fluorescent molecules comprise one or more of Cy3 and Cy 5;
the probes comprise a telomere DNA probe and a centromere DNA probe, the sequence of the telomere DNA probe comprises 3 repeating units, and the sequence of the repeating units is shown as SEQ ID NO. 1.
7. The probe for telomere and centromere super-resolution imaging of claim 6, wherein: the sequence of the telomere DNA probe is shown as SEQ ID NO.2, and the sequence of the centromere DNA probe is shown as SEQ ID NO. 3.
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