A method for imaging magnesium ions in living cells based on chemically modified deoxyribozymes

Abstract

The application discloses a method for imaging magnesium ions in living cells based on chemically modified deoxyribozyme, and belongs to the technical field of metal ion imaging. The method comprises the following steps: chemically modifying deoxyribozyme 10-23, screening and obtaining deoxyribozyme 10-23 (CaBn) which is modified with carboxyl (Ca) at the 8th position of the catalytic ring and benzene ring (Bn) at the 12th position; performing biochemical characterization and comparison; constructing a fluorescence sensor responding to magnesium ions; characterizing the performance; and applying the fluorescence sensor to imaging magnesium ions in living cells. Compared with the wild type, the application can catalyze the cleavage reaction under the condition of lower concentration of magnesium ions; therefore, the fluorescence sensor constructed from the deoxyribozyme can more effectively respond to lower concentration of magnesium ions in vitro, and the detection limit reaches 0.03 mM; in addition, the fluorescence sensor can perform fluorescence imaging on endogenous magnesium ions in living cells, and provides a new molecular tool for the field of molecular biology.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of metal ion imaging, and relates to a method for detecting magnesium ions in living cells based on chemically modified deoxyribozyme 10-23, in particular to a method for applying chemically modified deoxyribozyme with greatly improved activity and higher sensitivity to magnesium ions to imaging endogenous magnesium ions in cells. BACKGROUND

[0002] Magnesium ions are the most abundant divalent metal cations in cells and are closely related to many physiological processes. For example, as a co-factor of many enzymes, magnesium ions participate in chemical reactions in the body, as a co-factor of ATP, magnesium ions participate in energy metabolism, and magnesium ions maintain the stability of cell membranes. In addition, studies have shown that the content of magnesium ions is also closely related to immune response and the formation of some diseases. Therefore, the field of detection and imaging of magnesium ions in cells has always been a research hotspot. However, since magnesium ions in cells are mostly combined with related proteins, the concentration of truly free magnesium ions is lower (~1 mM), and its detection and imaging also face challenges. Most of the current methods for determining free magnesium ions in cells will cause certain damage and destruction to the cells, and cannot achieve non-destructive imaging of living cells, such as ion-selective electrode method.

[0003] Deoxyribozyme is a kind of DNA with catalytic ability obtained by in vitro screening. For example, deoxyribozyme 10-23 can catalyze the cleavage reaction of RNA substrate under the condition of the presence of specific divalent metal ions (such as magnesium ions). This process is more mild and less toxic, and in addition to the biocompatibility and low immunogenicity of deoxyribozyme itself, it has the potential to be applied to magnesium ion imaging. However, due to the instability of deoxyribozyme and its insufficient sensitivity to magnesium ions, it needs to be in a high concentration of magnesium ions to catalyze the reaction to occur, so its development in the field of magnesium ion imaging is limited. In view of this problem, the application provides a method for imaging magnesium ions in living cells based on chemically modified deoxyribozyme (10-23). SUMMARY

[0004] In view of the above problems, the application provides a method for imaging magnesium ions in living cells based on chemically modified deoxyribozyme (10-23), in particular to a method for applying chemically modified deoxyribozyme with greatly improved activity and higher sensitivity to magnesium ions to imaging endogenous magnesium ions in cells.

[0005] The technical scheme of the application is as follows: the method for imaging magnesium ions in cells based on chemically modified deoxyribozyme provided by the application has the following specific operation steps:

[0006] Step (1), the method for modifying DNA by using glycosidase and oxylamine compound to modify deoxyribozyme 10-23, the catalytic activity of the deoxyribozyme 10-23 (CaBn) with the highest catalytic activity is screened by introducing different chemical modifications at different positions of the catalytic loop;

[0007] Step (2), the biochemistry characterization of the deoxyribozyme 10-23 (CaBn) with the improved activity after chemical modification;

[0008] Step (3), the chemical modified deoxyribozyme 10-23 (CaBn) is constructed into a fluorescence sensor responding to magnesium ions;

[0009] Step (4), the performance of the constructed fluorescence sensor is characterized in vitro buffer, including sensitivity and specificity;

[0010] Step (5), the above fluorescence sensor is applied to the imaging of intracellular magnesium ions in living cells.

[0011] Further, in step (1), the specific process for obtaining the deoxyribozyme 10-23 with the highest catalytic activity includes:

[0012] First, the atypical bases (hypoxanthine and uracil) are introduced at different positions of the catalytic loop of the deoxyribozyme 10-23 by using the method of solid-phase synthesis;

[0013] Then, the alkylating adenine DNA glycosidase or uracil DNA glycosidase is added to react with the DNA double strand or single strand in the corresponding buffer solution, the atypical bases are removed to generate the abasic site;

[0014] Then, the glycosidase and the DNA single strand or double strand are separated by using the method of ethanol precipitation, and the oxylamine compound with different functional groups is added in the corresponding buffer solution to react, and the corresponding chemical modification is introduced at the abasic site;

[0015] Then, the modified single strand is purified by the method of ethanol precipitation or 3k ultrafiltration tube desalting, or the biotin-labeled DNA double strand is separated by using the streptavidin separation method to obtain the DNA single strand with modification;

[0016] Finally, the deoxyribozyme 10-23 with chemical modification obtained above is tested for activity under the same conditions (pH 7.5, 1mM Mg 2+ ) to screen the deoxyribozyme with the highest catalytic activity for subsequent construction of the fluorescence sensor;

[0017] The deoxyribozyme with the most improved catalytic activity is a double-modified deoxyribozyme 10-23(CaBn) in which the 8th position of the catalytic loop is modified with a carboxyl group (Ca) and the 12th position is modified with a benzene ring (Bn).

[0018] Further, in step (2), the specific process of biochemically characterizing the deoxyribozyme 10-23(CaBn) with improved activity after chemical modification includes:

[0019] First, the first-order reaction rate constant of the double-modified deoxyribozyme 10-23(CaBn) was tested under single turnover conditions;

[0020] Next, the Michaelis constant and catalytic constant of the double-modified deoxyribozyme 10-23(CaBn) were tested under multiple turnover conditions;

[0021] Finally, different concentrations of magnesium ions were added to the reaction solution, and the concentration response of the double-modified deoxyribozyme to magnesium ions was tested.

[0022] Further, in step (3), the specific process of constructing the deoxyribozyme 10-23 modified by chemical modification into a fluorescent sensor responsive to magnesium ions includes:

[0023] First, a solid-phase synthesis method was used to modify the 5' end and 3' end of the RNA substrate corresponding to the deoxyribozyme with a fluorescent group 6-carboxyfluorescein (FAM) and a corresponding quenching group BHQ-1, respectively;

[0024] Then, the above substrate and double-modified deoxyribozyme were added to pure water at a ratio of 1:1, and annealing was performed on a PCR instrument (85℃ for 5min, 4℃ for 5min, and 7℃ for 1min) to obtain a fluorescent sensor that can respond to magnesium ions;

[0025] This fluorescent sensor was incubated with a buffer solution. When magnesium ions were present in the solution, the deoxyribozyme cut the RNA substrate, causing the fluorescent group to separate from the quenching group, and the fluorescence signal to increase. When there were no magnesium ions in the solution, the fluorescence signal intensity was stable.

[0026] Further, in step (4), the specific process of characterizing the performance of the constructed fluorescent sensor in an in vitro buffer solution includes:

[0027] First, several divalent metal ions present in cells were added to the reaction buffer at their respective physiological concentrations in cells. Under the same conditions, the cutting efficiency of the double-modified deoxyribozyme on the substrate was tested by fluorescence intensity changes using an enzyme marker instrument to demonstrate its selectivity for magnesium ions.

[0028] Then, the fluorescence signal intensity of the sensor under different concentrations of magnesium ions is tested by an enzyme label instrument. When the concentration of magnesium ions is less than 2mM, the fluorescence signal intensity of the reaction system shows a linear relationship with the concentration of magnesium ions, and the detection limit is calculated as 0.03mM according to the formula of 3sigma / slope.

[0029] Further, in step (5), the specific process of applying the above-mentioned fluorescent sensor to the imaging of intracellular magnesium ions in living cells comprises:

[0030] Firstly, the constructed fluorescent sensor is encapsulated and transfected into the cell interior by using transfection reagent Lipofectamine 3000;

[0031] Then, the transfected cells are cultured in a cell incubator at 37 DEG C for 4h;

[0032] Finally, after the cell is fixed, the cell is imaged by using a laser scanning confocal microscope;

[0033] The performance is as follows: the fluorescent sensor can realize the imaging of endogenous magnesium ions in cells.

[0034] The beneficial effects of the present application are: by performing chemical modification on different sites and different functional groups of the catalytic ring part of the deoxyribozyme 10-23, a double-modified deoxyribozyme (CaBn) with greatly improved catalytic activity is finally obtained; the enzyme has high specificity for magnesium ions and can respond to lower concentrations of magnesium ions in a shorter time; after being constructed into a fluorescent sensor, it not only realizes the gradient response to different magnesium ion concentrations in vitro buffer, and the detection limit reaches 0.03mM; after being transfected into cells, it also successfully realizes the imaging of endogenous magnesium ions in cells; this application provides a good application prospect of molecular tools for the field of biological sensing, especially the field of intracellular magnesium ion imaging. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is a schematic diagram for testing the activity of the deoxyribozyme 10-23 modified by different chemicals in the present application;

[0036] Figure 2 It is a comparison diagram of the biochemical characterization results of the double-modified deoxyribozyme 10-23 (CaBn) screened in the present application;

[0037] Figure 3 It is a design schematic diagram of the magnesium ion fluorescent sensor constructed in the present application;

[0038] Figure 4 It is a comparison diagram of the in vitro performance characterization results of the magnesium ion fluorescent sensor constructed in the present application;

[0039] Figure 5Results of the present application based on chemically modified deoxyribozyme 10-23 (CaBn) for imaging endogenous magnesium ions in living cells. DETAILED DESCRIPTION

[0040] In order to more clearly illustrate the technical solutions of the present application, the technical solutions of the present application will be further described in detail below in combination with the accompanying drawings:

[0041] Example 1 as shown in Figure 1 The chemically modified deoxyribozyme 10-23 (CaBn) with the highest catalytic activity was obtained by screening:

[0042] 1. The method of modifying DNA with glycosidase and oxylamine compound (for details, refer to the published patent: CN112920247 A) was used to introduce different chemical modifications at different sites in the catalytic loop of deoxyribozyme 10-23;

[0043] 2. The different chemically modified deoxyribozyme 10-23 obtained above was dissolved in pure water and then added to an EP tube. Subsequently, a DNA substrate with two embedded RNAs was added, so that the ratio of enzyme to substrate was 40:1 (the final concentrations were 2mM and 50nM, respectively); annealing was performed on a PCR instrument (5min at 85℃, direct cooling to 4℃ for 5min, then recovery to 37℃ for 1min);

[0044] 3. Reaction buffer containing magnesium ions (1mM MgCl2, 20mM Tris-HCl, 100mM NaCl, pH 7.5) was added, and after 20s of reaction at 37℃, 2 times the volume of stop buffer (7M urea, 20mM EDTA, pH 8.0) was added to stop the reaction;

[0045] 4. The above reaction was characterized using 20% denaturing polyacrylamide gel electrophoresis, and after electrophoresis at 3000V, 100W for 1h, imaging was performed using an Odyssey CLx dual-color infrared laser imaging system (700nm excitation channel);

[0046] 5. The reaction yield was calculated according to the gray value of the product and substrate bands, and the catalytic activity of the different chemically modified deoxyribozymes was compared;

[0047] 6. Finally, it was determined that the double-modified 10-23 (CaBn) with carboxyl modification at position 8 and benzene ring modification at position 12 had the highest catalytic activity; under single turnover conditions, its first-order reaction rate constant was 800 times that of the unmodified deoxyribozyme 10-23.

[0048] Example 2 The process of using the magnesium ion fluorescent sensor constructed in the present application for intracellular magnesium ion imaging:

[0049] 1. Constructing CaBn-based magnesium ion fluorescent sensor

[0050] 1.1. Synthesis of sequences used: deoxyribozyme 10-23 DNA sequence with atypical base: 5'-AGGGTTGAGGCTAGCUACAHxCGATCATCTGT-3'; complementary strand sequence with biotin modification: 5'-Biotin-TTTAACTCTGATCGTTGTAGCTAGCC-3'; substrate with fluorescent group and quenching group: 5'-FAM-ACAGATGArGrUCAACCCT-BHQ-1-3';

[0051] 1.2. According to the published patent "A method for modifying DNA with glycosylase and oxylamine compound (for specific process, refer to the published patent)", alkylating adenine DNA glycosylase and uracil DNA glycosylase combined with oxylamine compound are used to introduce carboxyl modification at position 8 of the catalytic loop of deoxyribozyme 10-23 and benzene ring modification at position 12, respectively, to obtain double-modified deoxyribozyme 10-23 (CaBn);

[0052] 1.3. The double-modified deoxyribozyme 10-23 (CaBn) and the substrate (100 pmol) prepared above are added in an EP tube at a ratio of 1:1, mixed, and then placed in a PCR instrument for annealing (5 min at 85°C, direct cooling to 4°C for 5 min, then recovery to 37°C for 1 min);

[0053] 2. In vitro performance characterization of the constructed magnesium ion fluorescent sensor

[0054] 2.1. Specificity determination: the constructed magnesium ion sensor is placed in a reaction buffer containing different divalent metal ions (the concentration of each divalent metal ion is selected as the physiological concentration in cells), and reacted at 37°C for 2 min; the intensity of the emission light at 525 nm is collected using an enzyme marker at an excitation wavelength of 484 nm; as shown in Figure 4 ;

[0055] 2.2. Sensitivity determination: the reaction buffer containing different concentrations of magnesium ions is added to the fluorescent sensor system, and reacted at 37°C for 2 min; then, the fluorescence spectrum is scanned and collected using an enzyme marker at an excitation wavelength of 460 nm, and the emission wavelength is 500-650 nm; the results are shown in Figure 4 ;

[0056] 3. In-cell imaging of the constructed magnesium ion fluorescent sensor

[0057] 3.1, Hela cell culture. Hela cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, 100 mg / mL streptomycin and 2.5 μg / mL tetracycline at 37 °C in a humidified incubator containing 5% CO2; before confocal imaging, cells were plated in 35 mm glass-bottomed dishes and grown for 24 h, at which time the cells in the field of view reached 60-70% confluence;

[0058] 3.2, 100 pmol of sensor and 3 μL of Lipofectamine 3000 were separately incubated in Opti-MEM medium at room temperature for 5 min, then mixed at room temperature for 10 min. The mixture was added to PBS and incubated with cells for 4 h;

[0059] 3.3, After 4 h of incubation, 2.5 ng / mL of Hoechst 33258 nuclear stain was used to stain the cells at 37 °C for 8 min; then, the cells were carefully washed several times with PBS; further, the cells were fixed by treating with 4% paraformaldehyde at 37 °C for 15 min; finally, the 4% paraformaldehyde was replaced with PBS;

[0060] The above-mentioned treated cells were photographed using an Olympus laser scanning confocal microscope at a resolution of 40x; the fluorescence emission of Hoechst 3325 was measured at 430-470 nm under excitation light at 405 nm; the fluorescence of FAM was collected at 500-600 nm under excitation light at 488 nm; the results are shown in Figure 5

[0061] Finally, it should be understood that the embodiments described herein are merely illustrative of the principles of the embodiments of the present application; other variations can also be possible within the scope of the present application; therefore, as an example but not limitation, alternative configurations of the embodiments of the present application can be considered consistent with the teachings of the present application; accordingly, the embodiments of the present application are not limited to the embodiments explicitly introduced and described in the present application.​

Claims

1. A method for imaging magnesium ions in living cells based on chemically modified deoxyribozymes, characterized by, The method comprises the following steps: Step (1), chemically modifying deoxyribozyme 10-23 by using a glycosidase and an oxylamine compound, and screening the deoxyribozyme 10-23 with the highest catalytic activity by introducing different chemical modifications at different positions of the catalytic loop; The double-modified deoxyribozyme with 800 times of catalytic activity of the unmodified deoxyribozyme is obtained by chemically modifying the deoxyribozyme 10-23, and the deoxyribozyme introduces a carboxyl group at the 8th position of the catalytic loop and a benzene ring at the 12th position; Step (2), biochemically characterizing the deoxyribozyme 10-23 with the improved activity after chemical modification; The measured first order rate constant for the bi-modified deoxyribozyme 10-23 under single turnover conditions is 4.21 min -1 ; and the catalytic constant under multiple turnover conditions is 1.6 min -1 ; Step (3), constructing a fluorescence sensor responding to magnesium ions by using the chemically modified deoxyribozyme 10-23; constructing a fluorescence sensor responding to magnesium ions by using the double-modified deoxyribozyme 10-23; the fluorescence sensor has stable fluorescence signal in the absence of magnesium ions and enhanced fluorescence signal in the presence of magnesium ions; Step (4), characterizing the performance of the constructed fluorescence sensor in an in-vitro buffer; the performance of the constructed fluorescence sensor in the in-vitro buffer includes sensitivity and specificity; The constructed fluorescent sensor has a detection limit of 0.03 mM under cell physiological conditions 2+ with a specific response Step (5), applying the above fluorescence sensor to imaging of intracellular magnesium ions in living cells; The endogenous magnesium ions in cells are imaged by the fluorescence sensor.

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