A G-quadruplex nanosheet and its preparation method and application

Abstract

The present invention discloses a G-quadruplex nanosheet, its preparation method and application. The G-quadruplex nanosheet uses single-stranded DNA as a structural unit, rationally attaches a G-quadruplex nucleotide sequence to the DNA nanosheet, and constructs a nanosheet structure enriched with G-quadruplexes. The construction method is to obtain 9 nucleotide units in a buffer solution through programmed temperature control. The G-quadruplex nanosheet can be used for the treatment of heavy metal Pb 2+ Detection.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the preparation and application of a G-quadruplex nanosheet. Background Art

[0002] The G-quadruplex structure is a highly folded, stable secondary structure formed by single-stranded DNA rich in guanine (G) bases under the influence of certain ions or molecules. G-quadruplexes bind to hemin to form DNA enzymes with peroxidase-like activity. These enzymes catalyze the oxidation of substrates (such as 3,3',5,5'-tetramethylbenzidine (TMB) with H2O2, producing a distinct color change and have been widely used in biosensing. However, the primary drawback of free DNA enzymes is their relatively low activity. Therefore, the design and construction of DNA enzymes with simple structures and high catalytic activity is of great significance.

[0003] The present invention enriches G-quadruplex nucleotide sequences through DNA self-assembly, constructs G-quadruplex nanosheet structures with the assistance of potassium ions, and combines with hemin to form G-quadruplex DNA enzyme. The G-quadruplex nanosheet effectively enriches multiple G-quadruplexes on a nanostructure, which can significantly improve the catalytic activity of G-quadruplexes. This new DNA enzyme has a simple synthesis method and high catalytic activity, and is a novel enzyme for heavy metal Pb 2+ Sensitive detection is possible. Summary of the Invention

[0004] The purpose of the present invention is to provide a G-quadruplex nanosheet and a preparation method and application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention first provides a G-quadruplex nanosheet, which is composed of 9 DNA chains, as shown in SEQ ID NOs. 1 to 9; among them, the 5' ends of 8 DNA chains (S1 to S8) are modified with G-quadruplex sequences, and one DNA chain (S9) is used to stabilize the DNA nanosheet structure.

[0007] The present invention also provides a method for synthesizing the above-mentioned G-quadruplex nanosheet, the steps of which are as follows: dissolving 9 DNA chains S1 to S9 in 1xTAE buffer at equimolar concentrations (0.5 μM-2 μM), heating at 95°C for 5 minutes, then cooling to 20°C at a rate of 1°C per minute, and maintaining at 20°C for 1 hour to obtain a G-quadruplex nanosheet structure.

[0008] The present invention also provides the above-mentioned G-quadruplex nanosheet on heavy metal Pb 2+Applications in ion detection.

[0009] Preferably: 20 μL of G-quadruplex nanosheets are mixed with 2 μL of KSCN solution (500 mM) and Pb(NO3)2 solution of different concentrations, and reacted at room temperature for 1 hour; then 2.5 μL of hemin (50 μM) is added, and the reaction is continued at room temperature for 1 hour, and finally 180 μL of a colorimetric solution containing TMB and H2O2 is added, and the reaction is continued at room temperature for half an hour; the absorbance of the solution is measured by a UV spectrophotometer, and the absorbances of the lead ion and blank solution are obtained as A and A0, respectively, and the absorbance change Q=A0-A is calculated. 2+ Will be with K + Competition leads to the loss of catalytic activity of DNA enzyme. 2+ As the concentration increases, the absorbance changes gradually become larger. 2+ Plot the corresponding absorbance change Q and draw the UV absorption spectrum.

[0010] Beneficial effects: (1) The present invention solves the disadvantage of low catalytic activity of traditional G-quadruplexes and provides a new and simple method for preparing G-quadruplex nanosheets; (2) G-quadruplex nanosheets bring multiple G-quadruplex structures closer to one plane, which can effectively improve the catalytic efficiency of G-quadruplexes and improve the detection sensitivity; (3) The examples show that using the same concentration of G-quadruplexes and G-quadruplex nanosheets as DNA nanozymes for the detection of pb 2+ , G-quadruplex nanosheets exhibit better catalytic activity and obtain more intuitive color contrast. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Schematic diagram of the structure of G-quadruplex nanosheet.

[0012] Figure 2 Verify the synthesis of G-quadruplex nanosheets by acrylamide gel imaging.

[0013] Figure 3 This is an imaging photograph of G-quadruplex detecting lead ions.

[0014] Figure 4 This is the UV absorption spectrum of G-quadruplex nanoparticles detecting lead ions.

[0015] Figure 5 Imaging photos of G-quadruplex nanosheets detecting lead ions. DETAILED DESCRIPTION

[0016] To make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings, but the present invention is not limited to the following embodiments.

[0017] The DNA sequence used in the experiment of the present invention is shown in Table 1.

[0018] Table 1 Complete nucleotide sequences used in the experiment

[0019]

[0020] Example 1

[0021] DNA chain S1 was dissolved in 1xTAE buffer to a final concentration of 2 μM, heated at 95°C for 5 minutes, then cooled to 20°C at a cooling rate of 1°C / min and incubated at 20°C for 1 hour. The resulting reaction solution was labeled reaction solution 1.

[0022] DNA chains S1 and S2 were dissolved together in 1xTAE buffer to a final concentration of 2 μM. The mixture was heated at 95°C for 5 minutes, then cooled to 20°C at a cooling rate of 1°C / min and incubated at 20°C for 1 hour. The resulting reaction solution was labeled reaction solution 2.

[0023] DNA chains S1-S3 were dissolved together in 1xTAE buffer to a final concentration of 2 μM. The mixture was heated at 95°C for 5 minutes, then cooled to 20°C at a cooling rate of 1°C / min and incubated at 20°C for 1 hour. The resulting reaction solution was labeled reaction solution 3.

[0024] DNA chains S1-S4 were dissolved together in 1xTAE buffer to a final concentration of 2 μM. The mixture was heated at 95°C for 5 minutes, then cooled to 20°C at a cooling rate of 1°C / min and incubated at 20°C for 1 hour. The resulting reaction solution was labeled reaction solution 4.

[0025] DNA chains S1-S5 were dissolved together in 1xTAE buffer to a final concentration of 2 μM. The mixture was heated at 95°C for 5 minutes, then cooled to 20°C at a cooling rate of 1°C / min and incubated at 20°C for 1 hour. The resulting reaction solution was labeled reaction solution 5.

[0026] DNA chains S1 to S6 were dissolved together in 1xTAE buffer to a final concentration of 2 μM. The mixture was heated at 95°C for 5 minutes, then cooled to 20°C at a cooling rate of 1°C / min and incubated at 20°C for 1 hour. The resulting reaction solution was labeled reaction solution 6.

[0027] DNA chains S1 to S7 were dissolved together in 1xTAE buffer to a final concentration of 2 μM. The mixture was heated at 95°C for 5 minutes, then cooled to 20°C at a cooling rate of 1°C / min and incubated at 20°C for 1 hour. The resulting reaction solution was labeled reaction solution 7.

[0028] DNA chains S1 to S8 were dissolved together in 1xTAE buffer to a final concentration of 2 μM. The mixture was heated at 95°C for 5 minutes, then cooled to 20°C at a cooling rate of 1°C / min and incubated at 20°C for 1 hour. The resulting reaction solution was labeled reaction solution 8.

[0029] The steps for synthesizing G-quadruplex nanosheets are as follows: DNA chains S1 to S9 are dissolved in 1xTAE buffer to a final concentration of 2 μM each, heated at 95°C for 5 minutes, then cooled to 20°C at a cooling rate of 1°C / min and incubated at 20°C for 1 hour to form a G-quadruplex nanosheet structure. The structure of the G-quadruplex nanosheet is shown in the figure. Figure 1 shown.

[0030] Example 2

[0031] The G-quadruplex nanosheets synthesized in Example 1 were characterized by polyacrylamide gel electrophoresis. Figure 2 As shown, lane 1 represents reaction solution 1 (S1 strand), lane 2 represents reaction solution 2 (S1 + S2), lane 3 represents reaction solution 3 (S1 + S2 + S3), lane 4 represents reaction solution 4 (S1 + S2 + S3 + S4), lane 5 represents reaction solution 5 (S1 + S2 + S3 + S4 + S5), lane 6 represents reaction solution 6 (S1 + S2 + S3 + S4 + S5 + S6), lane 7 represents reaction solution 7 (S1 + S2 + S3 + S4 + S5 + S6 + S7), lane 8 represents reaction solution 8 (S1 + S2 + S3 + S4 + S5 + S6 + S7 + S8), and lane 9 represents a G-quadruplex nanosheet. In lanes 1 to 9, as the number of DNA strands added increases, the gel runs become slower and the molecular weight increases, demonstrating the successful synthesis of the G-quadruplex nanosheets of the present invention.

[0032] Example 3

[0033] G-quadruplex DNA molecules were used for pb 2+The detection method is as follows: 20 μL of 1 μM G-quadruplex DNA molecules (the G-quadruplex DNA molecules are single-stranded, and their nucleotide sequence is 5'-GGGTAGGGCGGGTTGGGT-3') are mixed with 2 μL of 500 mM KSCN solution and 20 μL of Pb(NO3)2 solutions of different concentrations (0 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1000 nM, 2000 nM, 5000 nM), and react at room temperature for 1 hour; then 2.5 μL of 50 μM hemin is added, and the reaction is continued at room temperature for 1 hour, and finally 180 μL of single-component TMB color development solution (purchased from Biyuntian Biotechnology Co., Ltd.) is added and reacted at room temperature for half an hour. The color changes of the experimental groups that cannot be detected are recorded using a smartphone. The results are as follows Figure 3 As shown in the figure, with the increase of the concentration of Pb(NO3)2 solution, the color of the solution gradually becomes darker after color development.

[0034] Example 4

[0035] The G-quadruplex nanosheets synthesized in Example 1 were used for 2+ The detection method is as follows: 20 μL of 1 μM G-quadruplex nanosheets are mixed with 2 μL of 500 mM KSCN solution and 20 μL of Pb(NO3)2 solution of different concentrations (0 nM, 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1000 nM, 2000 nM, 5000 nM) and reacted at room temperature for 1 hour; then 2.5 μL of 50 μM hemin was added and the reaction continued at room temperature for 1 hour. Finally, 180 μL of single-component TMB colorimetric solution (purchased from Biyuntian Biotechnology Co., Ltd.) was added and reacted at room temperature for half an hour. The absorbance of the solution was measured by UV spectrophotometer, and the absorbance of lead ions and blank control solution was obtained as A and A0, respectively. The absorbance change Q=A0-A was calculated. 2+ Will be with K + Competition leads to the loss of catalytic activity of DNA enzyme. 2+ As the concentration increases, the absorbance changes gradually become larger. 2+ Plot the corresponding absorbance change Q and draw the UV absorption spectrum. Figure 4 As shown in Figure 2, the UV absorption intensity increases with the increase of the concentration of Pb(NO3)2 solution. The color changes of different experimental groups were recorded by smartphone. Figure 5 As shown in the figure, with the increase of the concentration of Pb(NO3)2 solution, the color of the solution gradually becomes darker after detection, and the degree of darkening is greater than Figure 3It is stronger, proving that G-quadruplex nanosheets have better detection effect than G-quadruplex DNA molecules.

[0036] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. SEQUENCE LISTING <110> Minjiang College <120> A G-quadruplex nanosheet and its preparation method and application <130> <160> 10 <170> PatentIn version 3.3 <210> 1 <211> 77 <212> DNA <213> Artificial sequence <400> 1 gggtagggcg ggttgggttt tttggttcat tgcggagttc agtcttagat ggatctcgga 60 tgcaaggcct tctctcg 77 <210> 2 <211> 82 <212> DNA <213> Artificial sequence <400> 2 gggtagggcg ggttgggttt ttttcagctg gcctatctaa gactgaactc gcaccgccgg 60 cataagctat gcgctctgcc gc 82 <210> 3 <211> 45 <212> DNA <213> Artificial sequence <400> 3 gggtagggcg ggttgggttt tttggcagca gttcaggcca gctga 45 <210> 4 <211> 66 <212> DNA <213> Artificial sequence <400> 4 gggtagggcg ggttgggttt tttcgagaga aggcttgcca ggttacgttc gtacatcgtc 60 tgagtt 66 <210> 5 <211> 67 <212> DNA <213> Artificial sequence <400> 5 gggtagggcg ggttgggttt ttttcgaccg agcgtgaatt agtgatccgg aactcgcgca 60 atgaacc 67 <210> 6 <211> 45 <212> DNA <213> Artificial sequence <400> 6 gggtagggcg ggttgggttt tttgcggcag agcgacgctc ggtcg 45 <210> 7 <211> 82 <212> DNA <213> Artificial sequence <400> 7 gggtagggcg ggttgggttt tttttaggag atggcacgtt aatgaatagt ctccacttgc 60 atccgagatc cgaactgctg cc 82 <210> 8 <211> 45 <212> DNA <213> Artificial sequence <400> 8 gggtagggcg ggttgggttt tttaactcag acgaccatct cctaa 45 <210> 9 <211> 84 <212> DNA <213> Artificial sequence <400> 9 ggtgccgagt tccggatcac taattccata gcttatgccg gcactattca ttaacgtgtg 60 tacgaacgta acctggcaat ggag 84 <210> 10 <211> 18 <212> DNA <213> Artificial sequence <400> 10 gggtagggcg ggttgggt 18

Claims

1. A method for detecting heavy metal ions Pb 2+ The G-quadruplex nanosheet is characterized by: The G-quadruplex nanosheet is composed of 9 DNA chains, and the nucleotide sequences of the 9 DNA chains are as follows: S1: 5'-GGGTAGGGCGGGTTGGGTTTTTTGGTTCATTGCGGAGTTCAGTCTTAGATGGATCTCGGATGCAAGGCCTTCTCTCG-3', S2: 5'-GGGTAGGGCGGGTTGGGTTTTTTTCAGCTGGCCTATCTAAGACTGAACTCGCACCGCCGGCATAAGCTATGCGCTCTGCCGC-3', S3: 5'-GGGTAGGGCGGGTTGGGTTTTTTGGCAGCAGTTCAGGCCAGCTGA-3', S4: 5'-GGGTAGGGCGGGTTGGGTTTTTTCGAGAGAAGGCTTGCCAGGTTACGTTCGTACATCGTCTGAGTT-3', S5: 5'-GGGTAGGGCGGGTTGGGTTTTTTTCGACCGAGCGTGAATTAGTGATCCGGAACTCGCGCAATGAACC-3', S6: 5'-GGGTAGGGCGGGTTGGGTTTTTTGCGGCAGAGCGACGCTCGGTCG-3', S7: 5'-GGGTAGGGCGGGTTGGGTTTTTTTTAGGAGATGGCACGTTAATGAATAGTCTCCACTTGCATCCGAGATCCGAACTGCTGCC-3', S8: 5'-GGGTAGGGCGGGTTGGGTTTTTTAACTCAGACGACCATTCCCTAA-3', S9: 5'-GGTGCCGAGTTCCGGATCACTAATTCCATAGCTTATGCCGGCACTATTCATTAACGTGTGTACGAACGTAACCTGGCAATGGAG-3'; The preparation steps of the G-quadruplex nanosheet are as follows: DNA chains S1~S9 are dissolved in 1×TAE buffer at an equimolar concentration of 0.5μM~2μM, heated at 95°C for 5 minutes, then cooled to 20°C at a cooling rate of 1°C / min and incubated at 20°C for 1 hour to form a G-quadruplex nanosheet structure.

2. The G-quadruplex nanosheet according to claim 1 is exposed to heavy metal ions Pb 2+ Application in detection.

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