Method for imaging intracellular nucleic acid based on chemically modified deoxyribozyme

Abstract

The invention discloses a method for imaging intracellular nucleic acid based on chemically modified deoxyribozyme. The method comprises the following operation steps: carrying out chemical functional group modification on each site of a catalytic ring of deoxyribozyme 8-17, and screening to obtain a deoxyribozyme 8-17 variant 11Bn which is subjected to benzyl modification on a No.11 site of the catalytic ring; carrying out biochemical characterization on the variants; by taking miRNA-21 as an example, a multi-component nuclease (MNAzyme) system is constructed based on a variant and is used for specifically detecting intracellular nucleic acid and characterizing the detection performance of the system; manganese dioxide nanosheets for delivering nucleic acid are synthesized, and characterization of material morphology, size and the like is carried out; and the probe is applied to imaging of intracellular nucleic acid in living cells. The nucleic acid sensor constructed on the basis of the modified variant can effectively perform fluorescence response at a lower concentration; in addition, the sensor constructed by the invention does not need a transfection reagent or signal amplification, can perform sensitive fluorescence imaging in living cells, and provides a new molecular tool for diagnosis and treatment of related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of intracellular RNA imaging and relates to a method for imaging intracellular nucleic acids based on chemically modified deoxyribozymes, and in particular to a method for applying a chemically modified deoxyribozyme variant with enhanced activity to imaging nucleic acids in living cells. This method can also be used for in vitro detection and imaging. Background Art

[0002] Nucleic acids (including DNA and RNA) are core biological molecules of life activities, carrying genetic information and participating in a wide range of regulatory functions; among them, functional RNA, such as microRNA (miRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), etc., play a vital role in gene expression regulation. Therefore, the efficient identification and accurate detection of specific nucleic acid molecules in cells is not only the basis for understanding their biological functions, but also the key to revealing complex life processes and disease mechanisms. However, because nucleic acid targets in cells generally have characteristics such as low abundance, short sequences, and high family homology, traditional detection methods such as reverse transcription polymerase chain reaction (RT-PCR), Northern blot hybridization, and in situ hybridization (ISH) have bottlenecks such as insufficient detection sensitivity, false positives, and complex operations.

[0003] Take miRNAs, for example. They are short, low-abundance endogenous non-coding RNAs that regulate gene expression by mediating messenger RNA (mRNA) degradation or inhibiting its translation. Studies have shown that aberrant miRNA expression is closely associated with a variety of pathological conditions, including metabolic diseases and malignancies (e.g., miRNA-21 expression is significantly upregulated in lung, hepatocellular, and breast cancer cells). Therefore, efficient identification and precise detection of miRNAs not only facilitates the understanding of intracellular RNA regulatory networks but also supports the development of disease diagnostic biomarkers and targeted therapeutic strategies. DNAzymes, a class of catalytically active DNA molecules obtained through in vitro screening, offer advantages such as small molecular weight, flexible design, ease of synthesis, and good biocompatibility, thus holding potential for intracellular nucleic acid detection. However, because DNA molecules contain only four bases—adenine, thymine, cytosine, and guanine—the paucity of inherent functional groups limits their catalytic activity, often requiring high target concentrations or signal amplification strategies to generate a detectable signal, hindering their application in intracellular trace nucleic acid detection. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for imaging intracellular nucleic acids based on chemically modified deoxyribozymes, which has enhanced activity after chemical modification and is more sensitive to target sequences.

[0005] Technical solution: The method of imaging intracellular nucleic acids based on chemically modified DNAzymes described in the present invention comprises the following steps:

[0006] (1) Chemically modifying various sites on the catalytic ring of deoxyribozyme 8-17 by a modification method using a combination of DNA glycosidase and an oxamine compound, testing the catalytic activity of the variants after introducing different functional groups, and screening for the deoxyribozyme 8-17 variant with the highest catalytic activity improvement;

[0007] (2) Biochemical characterization of the DNAzyme 8-17 variant 11Bn with the highest catalytic activity;

[0008] (3) Taking miRNA-21 as an example, an MNAzyme sensing system for specific detection of miRNA-21 was constructed based on the DNAzyme 8-17 variant with the highest activity improvement, and its detection performance was characterized in an in vitro buffer environment;

[0009] (4) Synthesize manganese dioxide nanosheets for nucleic acid delivery and characterize the material morphology and size;

[0010] (5) The above-mentioned variant 11Bn-based manganese dioxide nanosheet-MNAzyme sensor was applied to imaging of intracellular miRNA-21 in living cells.

[0011] Furthermore, in step (1), a variant of deoxyribozyme 8-17 with a catalytic activity about 7 times higher than that of unmodified deoxyribozyme 8-17 was obtained by performing various functional group modifications on various sites of the catalytic loop of the deoxyribozyme 8-17 and screening. The variant introduced a benzyl group at site 11 of the catalytic loop of the deoxyribozyme 8-17. The specific steps include:

[0012] First, the atypical base uracil was introduced into each site of the catalytic loop of deoxyribozyme 8-17 by solid-phase synthesis;

[0013] Next, uracil DNA glycosidase is used to react with the uracil-introduced single-stranded DNAzyme 8-17 in a buffer solution to remove the uracil and generate a base-deficient site.

[0014] Next, the glycosidase and the deoxyribozyme 8-17 with the base-deficient site are separated by ethanol precipitation, and amine compounds with different functional groups are added to react in the corresponding buffer, thereby introducing the corresponding chemical modification at the base-deficient site.

[0015] Finally, the modified DNAzyme 8-17 variant was obtained after ethanol precipitation. The variant and wild-type 8-17 were reacted under the same reaction conditions (pH 7.5, 25 mM MgCl2 2+) were used for activity testing to screen out the DNAzyme 8-17 variant with the highest catalytic activity improvement for the subsequent construction of an MNAzyme sensor for specific detection of miRNA-21; among them, the DNAzyme 8-17 variant with the highest catalytic activity improvement obtained after screening had a benzyl (Bn) modification at position 11 of the catalytic ring and was therefore named 11Bn.

[0016] Furthermore, in step (2), the first-order reaction rate constant of the deoxyribozyme 8-17 variant for the chimeric RNA substrate in single turnover and the catalytic constant in multiple turnover were measured. The first-order reaction rate constant for the chimeric RNA substrate in the physiological concentration of magnesium ions was 2.26h. -1 The multi-turnover catalytic constant is 11.6h -1 The specific steps include:

[0017] First, the metal ion responsiveness of the DNAzyme 8-17 variant 11Bn to the chimeric RNA substrate was tested under the condition of 1 mM metal ion concentration;

[0018] Next, the first-order reaction rate constant of the DNAzyme 8-17 variant 11Bn towards the chimeric RNA substrate was tested under single-turnover conditions;

[0019] Finally, under multiple turnover conditions, the Michaelis-Menten constant and catalytic constant of the DNAzyme 8-17 variant 11Bn for chimeric RNA substrates were tested under physiological concentrations of magnesium ions.

[0020] Furthermore, in step (3), miRNA-21 was used as an example, and an MNAzyme sensing system for specific detection of target sequences was constructed based on the DNAzyme 8-17 variant with the highest activity improvement. The sensing system only produces a significant fluorescent signal after the addition of miRNA-21, and has a specific response to the target detection object; the detection limit of the MNAzyme constructed based on the variant for miRNA-21 is 6.35 nM under physiological concentration of magnesium ions; it specifically includes:

[0021] First, a chimeric RNA substrate complementary to the sequence of the deoxyribozyme 8-17 variant 11Bn was synthesized through solid-phase synthesis. The fluorescent group 6-carboxyfluorescein (FAM) was modified at the T base 10 of its 5' end, and the corresponding quencher group BHQ-1 was modified at the T base 9 of its 3' end. The deoxyribozyme 8-17 was split into two chains between the catalytic loops C7 and G8, and binding arms partially complementary to the miRNA-21 sequence were added to each chain. A uracil modification was also added at position 11 of the original catalytic loop.

[0022] Next, the portion of the DNA chain with uracil modification is chemically functionalized by the method of step (1) to introduce a benzyl group at position 11 in the assembled MNAzyme catalytic ring;

[0023] Next, the chains obtained by solid-phase synthesis and chemical modification (where the ratio of the two DNA chains is 1:1 and the ratio of substrate to DNA is 2:1) are incubated with buffer at 37°C. When miRNA-21 is added to the solution, the DNA and the target detection object assemble to form an MNAzyme structure through base complementary pairing, and the DNA enzyme catalytic ring is intact, thereby cleaving the substrate, separating the fluorescent group from the quenching group, and releasing a fluorescent signal. Without the addition of miRNA-21, the catalytic ring separates, the substrate cannot be cleaved, and no fluorescent signal is generated.

[0024] Subsequently, using miRNA-21 sequences with one base mutation, miRNA-21 sequences with two base mutations, and miRNA-155, miRNA-122, and miRNA-375 sequences as controls, the fluorescence signal intensity was measured on a microplate reader to reflect the cleavage activity of the MNAzyme sensor system for miRNA-21 and the control group, thereby verifying the specificity of the sensor system for miRNA-21.

[0025] Finally, a microplate reader was used to test the changes in fluorescence intensity generated by MNAzymes constructed based on the deoxyribozyme 8-17 variant 11Bn and the deoxyribozyme 8-17 wild type in response to different concentrations of miRNA-21 under physiological magnesium ion conditions. For 11Bn, when the miRNA-21 concentration was lower than 40nM, the fluorescence intensity of the reaction system was linearly related to the miRNA-21 concentration, and its detection limit was 6.35nM calculated according to the 3×σ / slope formula. For the wild type, when the miRNA-21 concentration was lower than 80nM, the fluorescence intensity of the reaction system was linearly related to the miRNA-21 concentration, and its detection limit was 23.22nM calculated according to the 3×σ / slope formula.

[0026] Furthermore, after the manganese dioxide nanosheets for nucleic acid delivery are synthesized in step (4), their morphology, size and composition are characterized by transmission electron microscopy, atomic force microscopy and ultraviolet-visible spectroscopy; in addition, the properties of the manganese dioxide nanosheets reduced by glutathione and their cytotoxicity are tested for application in intracellular imaging; which specifically includes:

[0027] First, a mixture of TMA·OH (0.6 M) and H2O2 (3 wt%) (20 mL) was reacted with a MnCl2·4H2O solution (10 mL, 0.3 M). After forming a dark brown suspension, the suspension was stirred at room temperature overnight, centrifuged at 2000 rpm / min for 10 minutes, washed three times with deionized water and methanol, and dried at 60°C. 5 mg of the dried crude product was dispersed in 10 mL of deionized water and sonicated to obtain manganese dioxide nanosheets.

[0028] Then, the morphology, size and composition of the manganese dioxide nanosheets were characterized by transmission electron microscopy, atomic force microscopy and ultraviolet-visible spectroscopy, proving the successful preparation of manganese dioxide nanosheets.

[0029] Next, glutathione was added to the manganese dioxide nanosheets, and their absorbance was tested by UV-visible spectroscopy to prove their ability to be reduced by glutathione.

[0030] Subsequently, manganese dioxide nanosheets and glutathione were added to the buffer of the MNAzyme system, and the fluorescence signal was measured using a microplate reader to verify that the manganese dioxide nanosheets were reduced to produce manganese ions, which acted as a cofactor to promote the enzyme's cleavage of the substrate.

[0031] Finally, the cytotoxicity of manganese dioxide nanosheets was tested using Cell Counting Kit-8.

[0032] Furthermore, the step (5) specifically includes:

[0033] First, nucleic acids and manganese dioxide nanosheets were co-incubated with cells in a 37°C incubator for 4 h;

[0034] Next, the cells were imaged using a laser scanning confocal microscope after staining the nuclei;

[0035] Next, the miRNA-21 inhibitor was transfected into the cells using the transfection reagent Mirus TranslT, and the nucleic acid and manganese dioxide nanosheets were co-incubated with the cells in a 37°C incubator for 4 h;

[0036] Finally, the cell nuclei were stained and the cells were imaged using a laser scanning confocal microscope. The sensor demonstrated the following performance: the sensor was able to image intracellular miRNA-21 and sensitively distinguish between miRNAs of different concentrations.

[0037] Beneficial Effects: Compared with the prior art, the present invention has the following features: By chemically modifying various functional groups at various sites of the catalytic loop of deoxyribozyme 8-17, the present invention screened and obtained the deoxyribozyme 8-17 variant 11Bn with the highest catalytic activity. This variant introduced a benzyl modification at site 11 of the catalytic loop, and its first-order reaction rate constant was increased by up to approximately 7 times compared to the unmodified deoxyribozyme 8-17. Taking miRNA-21 as an example, the MNAzyme sensor system constructed based on this variant for the specific detection of intracellular nucleic acids has a detection limit of up to 6.35nM for the target analyte under physiological magnesium ion concentration conditions. In addition, the combined use of manganese dioxide nanosheets enables the sensor system to be delivered into cells without transfection reagents, successfully achieving sensitive imaging of trace nucleic acids in cells. This invention provides a simple and sensitive molecular tool for the field of biosensing, especially the field of intracellular nucleic acid imaging, and the invention can also be used for in vitro imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Graphs showing the results of testing the catalytic activity of variants after chemical modification of various sites in the catalytic loop of deoxyribozyme 8-17 and the biochemical characterization of the variants;

[0039] Figure 2 This is the design and imaging process of the MNAzyme sensor for specific detection of target sequences constructed based on the DNAzyme 8-17 variant 11Bn of the present invention;

[0040] Figure 3 Schematic diagram of the MNAzyme sensor constructed using miRNA-21 as an example and the characterization results in the present invention;

[0041] Figure 4 Schematic diagram of the characterization results of the manganese dioxide nanosheets synthesized for nucleic acid delivery in the present invention;

[0042] Figure 5 This is a schematic diagram of the results of the present invention achieving miRNA-21 imaging in living cells based on the deoxyribozyme 8-17 variant 11Bn. DETAILED DESCRIPTION

[0043] The present invention is further described below with reference to the accompanying drawings and specific embodiments.

[0044] Example 1

[0045] like Figure 1 As shown, after chemical modification of the catalytic ring of deoxyribozyme 8-17, the variant 11Bn with the highest catalytic activity was obtained through activity testing.

[0046] 1. By combining DNA glycosidase and amine compounds (refer to the published patent: CN112920247A for the specific process), different chemical functional groups were introduced into various sites of the catalytic loop of the deoxyribozyme 8-17;

[0047] 2. Dissolve the modified DNAzyme 8-17 variants obtained above in pure water and add them to separate EP tubes. Then, add a chimeric substrate with an RNA at the cleavage site so that the enzyme-to-substrate ratio in the EP tube is 10:1 (the final concentration of the enzyme is 500 nM, and the final concentration of the substrate is 50 nM). After mixing, anneal the tubes in a PCR instrument (incubate at 85°C for 5 minutes, then cool directly to 4°C for 5 minutes, and finally hold at 37°C for 1 minute).

[0048] 3. Centrifuge to precipitate water vapor in the tube, then add reaction buffer (25 mM MgCl2, 50 mM HEPES, 200 mM NaCl, pH 7.5). Incubate at 37°C for 30 s, then add 2 volumes of stop buffer (7 M urea, 20 mM EDTA, pH 8.0) to stop the reaction.

[0049] 4. The reaction was characterized using 20% ​​denaturing polyacrylamide gel electrophoresis at 3000V, 100W for 45 minutes, followed by imaging using an Odyssey CLx dual-color infrared laser imaging system (700nm excitation channel). The reaction yield was then calculated based on the grayscale values ​​of the product and substrate bands. The catalytic activity of the deoxyribozyme 8-17 variants with different chemical modifications was compared. Ultimately, the deoxyribozyme 8-17 variant 11Bn, with a benzyl modification at position 11 of the catalytic loop, was found to have the highest catalytic activity.

[0050] 5. Add a chimeric substrate containing an RNA at the cleavage site to the DNAzyme 8-17 variant 11Bn; after mixing and annealing, add reaction buffer containing different metal ions and react at 37°C. Stop the reaction with 2 volumes of stop buffer, and then identify the cleavage yield by gel electrophoresis to perform biochemical characterization of 11Bn, including metal ion responsiveness and first-order reaction rate constant, for example. Figure 2 As shown in Figure 2, the first-order reaction rate constant of 11Bn in 1 mM Mg is 2+ Under these conditions, the activity was three times that of unmodified DNAzyme 8-17.

[0051] Example 2

[0052] The present invention uses miRNA-21 as an example to construct an MNAzyme fluorescent sensor based on a chemically modified variant of DNAzyme 8-17 for intracellular nucleic acid imaging.

[0053] 1. Taking miRNA-21 as an example, an MNAzyme system for specific detection of intracellular nucleic acids was constructed based on 11Bn:

[0054] 1.1. Sequences used in synthesis:

[0055] Chimeric RNA substrates with fluorescent groups and quenching groups:

[0056] 5'-AGACGATCC / i6FAMdT / TrGGCT / iBHQ1dT / CTCATGCG-3';

[0057] A portion of the DNA sequence that separates the catalytic loop of DNAzyme 8-17 from C7:

[0058] 5'-CGCATGAGAAG TGTCAGC CTGATAAGCTA-3';

[0059] Another DNA sequence that separates DNAzyme 8-17 from G8 and carries a uracil modification:

[0060] 5'-TCAACATCAGT GACrUCGAA AAGGATCGTCT-3'; (the underlined portion is the catalytic loop sequence of the original DNAzyme 8-17);

[0061] miRNA-21 sequence: 5'-rUrArGrCrUrUrArUrCrArGrArCrUrGrArUrGrUrUrGrA-3';

[0062] miRNA-21 sequence with a single mismatch:

[0063] 5'-rUrArGrCrUrUrArUrCrArGrArCrUrGrArUrGrArUrGrA-3';

[0064] miRNA-21 sequence with two base mismatches:

[0065] 5'-rUrArGrCrUrUrArUrCrArGrArCrUrGrArUrGrArUrCrA-3';

[0066] miRNA-155 sequence: 5'-rUrUrArArUrGrCrUrArArUrCrGrUrGrArUrArGrGrGrGrU-3';

[0067] miRNA-122 sequence: 5'-rUrGrGrArGrUrGrUrGrArCrArArUrGrGrUrGrUrUrUrG-3';

[0068] miRNA-375 sequence: 5'-rUrUrUrGrUrUrCrGrUrUrCrGrGrCrUrCrGrCrGrUrGrA-3';

[0069] 1.2. Introducing benzyl modification into DNA sequences with uracil modification by combining DNA glycosidase and oxamine compounds (refer to the published patent CN112920247 A for the specific process);

[0070] 1.3. Mix the substrate and two DNA sequences in an EP tube at a 1:1 ratio between the two DNA strands and a 2:1 ratio between the substrate and DNA strands (final concentrations of 200 nM for both DNA strands and 400 nM for the substrate). Add reaction buffer (1 mM MgCl2, 50 mM HEPES, 200 mM NaCl, pH 7.5) and incubate at 37°C. Initiate the reaction by adding the miRNA-21 sequence.

[0071] 2. If Figure 3 The detection performance of the MNAzyme system shown is as follows:

[0072] 2.1. Specificity Assay: To the above reaction system without the addition of the miRNA-21 sequence, the miRNA-21 sequence, the miRNA-21 sequence with a single base mismatch, the miRNA-21 sequence with two base mismatches, the miRNA-122 sequence, the miRNA-155 sequence, and the miRNA-375 sequence were added, respectively, so that the ratio of miRNA to DNA strand in the solution was 1:1; incubated at 37°C for 4 h, and the intensity of the emitted light at 520 nm under an excitation wavelength of 480 nm was measured using a microplate reader;

[0073] 2.2 Sensitivity measurement: Different concentrations of miRNA-21 sequences were added to the above reaction system without the addition of miRNA-21 sequences. The mixture was incubated at 37°C for 4 h. The intensity of the emitted light at 520 nm under an excitation wavelength of 480 nm was measured using a microplate reader.

[0074] 3. If Figure 4 The synthesized manganese dioxide nanosheets were characterized as shown:

[0075] 3.1. Material Characterization: The morphology, size, and composition of the manganese dioxide nanosheets were characterized by transmission electron microscopy, atomic force microscopy, and UV-visible spectroscopy.

[0076] 3.2 Characterization of the Reduction Properties of Nanosheets: The miRNA-21 sequence was added to the above-mentioned MNAzyme reaction system to achieve a 1:1 ratio of miRNA to DNA strands in the solution. Appropriate amounts of manganese dioxide nanosheets and glutathione solution were also added. The mixture was incubated at 37°C for 4 h. The intensity of the emitted light at 520 nm under an excitation wavelength of 480 nm was measured using a microplate reader.

[0077] 3.3. Nanosheet Cytotoxicity Assay: MDA-MB-231 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 mg / mL streptomycin in a 37°C incubator containing 5% CO2. The cells were seeded into 96-well plates containing 100 μL of culture medium and cultured overnight. The cells were treated with various concentrations of manganese dioxide nanosheet solutions for 48 h. 10 μL of Cell Counting Kit-8 was then added to each well and incubated at 37°C for 2 h. Finally, cell proliferation was determined by measuring absorbance at 450 nm using a SpectraMax iD3 microplate reader.

[0078] 4. If Figure 5 The intracellular imaging results of the miRNA-21 fluorescent sensor constructed based on the DNAzyme 8-17 variant 11Bn are shown:

[0079] 4.1 Cell Culture: MDA-MB-231 and MCF10A cells were cultured in a 37°C incubator with 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 mg / mL streptomycin. Before confocal imaging, cells were plated in 35 mm glass-bottomed culture dishes and grown for 24 h, at which point the cells in the field of view reached 60-70% confluence.

[0080] 4.2. 200 pmol of DNA strand and 400 pmol of substrate were mixed with the manganese dioxide nanosheet solution and incubated in Opti-MEM medium for 5 min. Cell culture medium was then added. The mixture was incubated at room temperature and then added to the cells for 4 h. Subsequently, the cells were stained with 2.5 ng / mL Hoechst 33258 nuclear stain at 37°C for 8 min. After careful washing with PBS several times, medium supplemented with 10% fetal bovine serum was added.

[0081] 4.3. The treated cells were imaged using an Olympus laser scanning confocal microscope at 60× resolution. Hoechst 3325 fluorescence emission was measured in the range of 430-470 nm under 455 nm excitation. FAM fluorescence was collected in the range of 500-600 nm under 517 nm excitation.

[0082] 4.4 Transfection of miRNA-21 inhibitors: 100 pmol, 200 pmol, and 400 pmol of the inhibitor were incubated with 4 μL of Mirus TranslT in Opti-MEM medium at room temperature for 10 min. The two were then mixed and incubated with the cells for 24 h. The cells were then aspirated and incubated with MNAzyme-manganese dioxide nanosheet solution as above, stained, and subjected to confocal imaging.

Claims

1. A method for imaging intracellular nucleic acids based on chemically modified DNAzymes, characterized in that: The steps are as follows: (1) Chemically modifying various sites on the catalytic ring of deoxyribozyme 8-17 by a modification method using a combination of DNA glycosidase and an oxamine compound, testing the catalytic activity of the variants after introducing different functional groups, and screening for the deoxyribozyme 8-17 variant with the highest catalytic activity improvement; (2) biochemical characterization of the DNAzyme 8-17 variants with the highest catalytic activity obtained through screening; (3) Using miRNA-21 as an example, an MNAzyme sensing system for specific target sequence detection was constructed based on the DNAzyme 8-17 variant with the highest activity enhancement, and its detection performance was characterized in an in vitro buffer environment; (4) Synthesize manganese dioxide nanosheets for nucleic acid delivery and characterize the material morphology, size, and composition; (5) The variant-based manganese dioxide nanosheet-MNAzyme sensor was applied to image intracellular nucleic acids in living cells.

2. The method for imaging intracellular nucleic acids based on chemically modified DNAzymes according to claim 1, characterized in that: In the step (1), a variant of 8-17 with a 7-fold increased catalytic activity was obtained by performing various functional group modifications on various sites of the catalytic loop of the deoxyribozyme 8-17 and screening. The variant had a benzyl group introduced into site 11 of the catalytic loop.

3. The method for imaging intracellular nucleic acids based on chemically modified DNAzymes according to claim 2, characterized in that: The operation steps of step (1) are: First, the atypical base uracil was introduced into each site of the catalytic loop of deoxyribozyme 8-17 by solid-phase synthesis; Next, uracil DNA glycosidase was used to react with the single-stranded DNAzyme 8-17 into which uracil was introduced in a buffer solution; Next, the glycosidase and the deoxyribozyme 8-17 with a base-deficient site are separated by ethanol precipitation, and oxygen-amine compounds with different functional groups are added to react in a corresponding buffer; Finally, after ethanol precipitation, the modified DNAzyme 8-17 variant was obtained. The variant and wild-type 8-17 were tested for activity under the same reaction conditions to screen out the DNAzyme 8-17 variant with the highest catalytic activity improvement.

4. The method for imaging intracellular nucleic acids based on chemically modified DNAzymes according to claim 1, characterized in that: In step (2), the first-order reaction rate constant of the DNAzyme 8-17 variant for the chimeric RNA substrate in single turnover and the catalytic constant in multiple turnover are measured; The single-turnover first-order reaction rate for the chimeric RNA substrate under physiological magnesium ion conditions is 2.26 h -1 The multi-turnover catalytic constant is 11.6h -1 .

5. The method for imaging intracellular nucleic acids based on chemically modified DNAzymes according to claim 4, characterized in that: The operation steps of step (2) are: First, the metal ion responsiveness of the DNAzyme 8-17 variant 11Bn to the chimeric RNA substrate was tested under the condition of 1 mM metal ion concentration; Next, the first-order reaction rate constant of the DNAzyme 8-17 variant 11Bn towards the chimeric RNA substrate was tested under single-turnover conditions; Finally, under multiple turnover conditions, the Michaelis-Menten constant and catalytic constant of the DNAzyme 8-17 variant 11Bn for chimeric RNA substrates were tested under physiological concentrations of magnesium ions.

6. The method for imaging intracellular nucleic acids based on chemically modified DNAzymes according to claim 1, characterized in that: In step (3), miRNA-21 is used as an example, and an MNAzyme sensing system for specific detection of target sequences is constructed based on the DNAzyme 8-17 variant with the highest activity improvement. The sensing system generates a fluorescent signal only after the target sequence is added, and has a specific response to the target sequence; the detection limit of the MNAzyme sensing system constructed based on the variant for miRNA-21 under physiological concentration of magnesium ions is 6.35nM.

7. The method for imaging intracellular nucleic acids based on chemically modified DNAzymes according to claim 6, characterized in that: The operation steps of step (3) are as follows: First, a chimeric RNA substrate complementary to the sequence of deoxyribozyme 8-17 variant 11Bn was obtained by solid-phase synthesis; Next, the portion of the DNA chain with uracil modification is modified with chemical functional groups; Next, the chains obtained by the solid phase synthesis and chemical modification were incubated with a buffer at 37°C; Subsequently, the fluorescence signal intensity was tested on a microplate reader to reflect the cleavage activity of the MNAzyme sensing system for miRNA-21 and the control group; Finally, a microplate reader was used to test the changes in fluorescence intensity of the MNAzyme constructed based on the DNAzyme 8-17 variant 11Bn and the DNAzyme 8-17 wild type in response to different concentrations of miRNA-21 under physiological magnesium ion conditions.

8. The method for imaging intracellular nucleic acids based on chemically modified DNAzymes according to claim 1, characterized in that: In the step (4), the morphology, size and composition of the sample are characterized by transmission electron microscopy, atomic force microscopy and ultraviolet-visible spectroscopy; Furthermore, the glutathione-reduced properties and cytotoxicity of MnO2 nanosheets were tested for their application in intracellular imaging.

9. The method for imaging intracellular nucleic acids based on chemically modified DNAzymes according to claim 8, characterized in that: The operation steps of step (4) are: First, a mixture of TMA·OH and H2O2 was reacted with MnCl2·4H2O solution to obtain manganese dioxide nanosheets; Then, the morphology, size and composition of the manganese dioxide nanosheets were characterized by transmission electron microscopy, atomic force microscopy and UV-visible spectroscopy; Next, glutathione was added to the manganese dioxide nanosheets, and its absorbance was tested by UV-visible spectroscopy; Subsequently, manganese dioxide nanosheets and glutathione were added to the buffer of the MNAzyme system, and their fluorescence signals were measured using a microplate reader. Finally, the cytotoxicity of manganese dioxide nanosheets was tested using Cell Counting Kit-8.

10. The method for imaging intracellular nucleic acids based on chemically modified DNAzymes according to claim 8, characterized in that: The operation steps of step (5) are as follows: First, nucleic acids and manganese dioxide nanosheets were incubated with cells in an incubator; Next, the cells were imaged using a laser scanning confocal microscope after staining the nuclei; Next, after transfecting the miRNA-21 inhibitor into the cells using the transfection reagent Mirus TranslT, the nucleic acid and manganese dioxide nanosheets were incubated with the cells in an incubator; Finally, the cells were imaged using laser scanning confocal microscopy after staining the nuclei.

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