Construction and application of multifunctional triangular DNA biosensor

Abstract

The invention provides a preparation method of a multifunctional triangular DNA biosensor. In the invention, a multifunctional triangular DNA biosensor is rapidly constructed by a three-functional DNA single-chain one-pot method to realize glutathione (GSH) response so as to detect early growth reaction factor-1 (EGr-1) mRNA and silence the expression of the EGr-1 mRNA. When only GSH exists, a disulfide bond in SS-L is reduced by GSH and broken, but no target Egr-1 exists, and DNAzyme on TSDz cannot be combined with the target to generate fluorescence; under the condition that only a target object Egr-1 exists and no GSH exists, the Egr-1 can be combined with the 5'end part on the TSDz, so that a small amount of fluorescence is recovered; when GSH and a target object Egr-1 exist at the same time, GSH cuts a disulfide bond to release part of binding sites, DNAzyme on TSDz can be replaced under the action of the target object Egr-1 to generate a large amount of fluorescence, meanwhile, Egr-1 is cut under the action of magnesium ions to realize gene silencing, the fluorescence intensity is enhanced along with the increase of the concentration of the target object, and the fluorescence intensity is enhanced along with the increase of the concentration of the target object. And a good linear relationship is shown at 0-1nM. The method disclosed by the invention has high sensitivity to detection of Egr-1, and the detection limit is 0.25 nM. The method is of great significance to prediction and treatment of cancers.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry detection, and specifically relates to the construction and application of a multifunctional triangular DNA biosensor for detecting early growth response factor-1 (Egr-1) mRNA and silencing its expression by glutathione (GSH) response. Background Art

[0002] At present, malignant tumors are intractable diseases that endanger human health due to the low selectivity of conventional chemotherapy, high cell resistance, side effects on healthy tissues, tumor recurrence / metastasis, etc. The tumor microenvironment (TME) is crucial for the occurrence and development of tumors. Therefore, making full use of the tumor microenvironment is of great significance for improving the accuracy of tumor marker detection and treating cancer.

[0003] Glutathione (GSH), as a non-protein thiol, plays an irreplaceable role in maintaining redox homeostasis in biological systems by eliminating reactive oxygen species (ROS). Cancer cells have higher abundance levels of ROS to enhance the demand for aerobic glycolysis, which logically leads to higher GSH content, thereby protecting cells from ROS-induced apoptosis. Moreover, in most studies, glutathione is used to cleave disulfide bonds. Early growth response-1 (Egr-1), as a multidomain protein, is associated with several important physiological processes, including development, metabolism, cell growth and proliferation. Egr-1 is an anti-cancer gene that monitors DNA damage conservation and promotes tumor cell apoptosis, and adjuvants can improve the anti-cancer efficiency of chemoradiotherapy. However, under harsh environments, Egr-1 expression levels are elevated to maintain tumor cell survival, proliferation, metastasis and angiogenesis as an oncogene. Identifying and utilizing the "enemy" role of Egr-1 in cancer can open up horizons for gene therapy of patients with malignant tumors. Therefore, in the present invention, three functional DNA single strands of DNAzyme, SS-L and Apt-DNA form a multifunctional triangular DNA biosensor, and the fluorescence of FAM in DNAzyme is quenched by BHQ1 in Apt-DNA, resulting in a low background signal. The designed multifunctional triangular DNA biosensor uses Apt-DNA to accurately target cancer cells, while GSH response performs highly selective and highly sensitive detection and gene silencing of the target Egr-1. The sensor also exhibits excellent enzyme stability and time stability, which is of great significance and certain practical value for the accurate identification of markers in tumors and the treatment of cancer. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a multifunctional triangular DNA biosensor, which contains an aptamer targeting cancer cells to accurately identify cancer cells, and can also achieve glutathione (GSH) response in tumor cells to detect early growth response factor-1 (Egr-1) mRNA and silence its expression.

[0005] The purpose of the present invention is achieved through the following technical solutions.

[0006] The construction and application of a multifunctional triangular DNA biosensor is characterized by the following synthesis steps:

[0007] DNAzyme (10 μL, 20 μM), SS-L and Apt-DNA (20 μL, 20 μM) were mixed evenly with 150 μL PBS buffer and incubated at 37°C for 20 min in one pot to obtain a multifunctional triangular DNA probe (named TSDz).

[0008] The working principle of the present invention is:

[0009] (1) The multifunctional triangular DNA biosensor consists of three functional DNA single strands, namely DNAzyme, SS-L, and Apt-DNA.

[0010] (2) In the absence of GSH and the target Egr-1 mRNA, the FAM modified on the 5' end of the DNAzyme and the BHQ1 on the 3' end of the Apt-DNA are close to each other, resulting in the quenching of the FAM fluorescence by BHQ1, resulting in a lower background signal.

[0011] (3) When only GSH is present, the disulfide bonds in SS-L are reduced and broken by GSH, but in the absence of the target Egr-1 mRNA, the DNAzyme on TSDz will not bind to the target to produce fluorescence.

[0012] (4) When only the target Egr-1 mRNA is present but GSH is absent, Egr-1 mRNA will bind to the 5' end of TSDz, resulting in a small increase in fluorescence.

[0013] (5) When GSH and the target Egr-1 mRNA are present at the same time, GSH cuts the disulfide bonds to release some binding sites. Under the action of the target Egr-1 mRNA, the DNAzyme on TSDz can be replaced to produce a large amount of fluorescence. At the same time, under the action of magnesium ions, Egr-1 mRNA will be cut to achieve gene silencing.

[0014] The sequences of DNAzyme, SS-L and Apt-DNA are:

[0015] DNAzyme: 5'-AACCGCGGCCAGGCTAGCTACAACGACCTGGACGA-3';

[0016] SS-L: 5'-TAGTGTATATGCGTTTTCGTC / iHS-SH / CAGGTCGTTGTTTCCGCGGTTTTTCGTATCTCAGAGGA-3';

[0017] Apt-DNA: 5'-ACGCATATACACTACGCAGTTGATCCTTTGGATACCCTGGTC CTCTGAGATACGA-3'.

[0018] The present invention is designed to detect early growth response factor-1 (Egr-1) mRNA and silence its expression by glutathione (GSH) response.

[0019] The fluorescence detection conditions were set as: excitation wavelength: 488 nm, excitation slit: 5 nm, emission slit: 5 nm, voltage: 800 V. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] [ Figure 1 ] Schematic diagram of the multifunctional triangular DNA biosensor that responds to GSH, Egr-1 separately, and GSH and Egr-1 simultaneously.

[0021] [ Figure 2 ] Gel electrophoresis verified the construction of the multifunctional triangular DNA biosensor (TSDz) (A) and the optimization of the ratio of Dz:SS-L:Apt-DNA (B), and fluorescence verified the optimization of the ratio of Dz:SS-L:Apt-DNA (C) and its normalized fluorescence (D).

[0022] [ Figure 3 ] Time-optimized fluorescence plots (A) and normalized fluorescence (B) of the construction of the multifunctional triangular DNA biosensor (TSDz), and feasibility analysis of TSDz fluorescence in response to GSH and Egr-1 (C).

[0023] [ Figure 4 ] Fluorescence spectrum (A) and fluorescence ratio (F / F0) spectrum (B) of the control probe (probe TDz without disulfide bond modification) of the multifunctional triangular DNA biosensor (TSDz) in response to different concentrations of Egr-1, and kinetic analysis of TDz response to different concentrations of Egr-1 (C).

[0024] [ Figure 5] Fluorescence spectra (A) and linear spectra (B) of the multifunctional triangular DNA biosensor (TSDz) in response to different concentrations of Egr-1, and kinetic analysis of TSDz in response to different concentrations of Egr-1 (C).

[0025] [ Figure 6 ] Investigation of the selectivity of the multifunctional triangular DNA biosensor (TSDz) for different mRNAs (A), and comparison of the performance of TSDz in response to Egr-1 in serum (1%) and PBS buffer (B).

[0026] [ Figure 7 ]Study on the deoxyribonuclease I (DNase I) stability of the multifunctional triangular DNA biosensor (TSDz) (A), and study on the temporal stability of TSDz (B).

[0027] [ Figure 8 ]The DNA sequence required for this experiment. Specific implementation plan

[0028] Here, the specific implementation scheme of the present invention is further described in detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but do not limit the application scope and extension of the present invention.

[0029] Example 1: Principle design of a multifunctional triangular DNA biosensor.

[0030] like Figure 1 As shown in the figure, the multifunctional triangular DNA biosensor is composed of three functional DNA single strands. DNAzyme is a DNA enzyme chain that can cut the target Egr-1; SS-L is modified with disulfide bonds and can respond to GSH; Apt-DNA contains the MUC1 aptamer sequence and can target tumor cells. TSDz was synthesized in one pot at 37°C, and the sequence complementary to Egr-1 contained in DNAzyme was locked by SS-L, and the FMA on DNAzyme was quenched by BHQ1, resulting in a low fluorescence signal. When only GSH is present, the disulfide bond in SS-L is reduced and broken by GSH, but without the target Egr-1, the DNAzyme on TSDz will not bind to the target to produce fluorescence; when only the target Egr-1 is present but no GSH is present, Egr-1 will bind to the 5' end of TSDz, resulting in a small amount of fluorescence recovery; when GSH and the target Egr-1 are present at the same time, GSH cuts the disulfide bond to release some binding sites, and under the action of the target Egr-1, the DNAzyme on TSDz can be replaced to produce a large amount of fluorescence. At the same time, under the action of magnesium ions, Egr-1 will be cut to silence the gene.

[0031] Example 2: Construction of a multifunctional triangular DNA biosensor and feasibility analysis of its response targets.

[0032] The construction of the multifunctional triangular DNA biosensor was verified by polyacrylamide gel electrophoresis experiments. Figure 2 Lanes 1-6 in A are DNAzyme, SS-L, Apt-DNA, DNAzyme+SS-L, DNAzyme+SS-L+Apt-DNA and SS-L+Apt-DNA, respectively. The new bands produced in lanes 4 and 6 are DNAzyme / SS-L and SS-L / Apt-DNA hybrid double-stranded bands, respectively; the new band appearing in lane 5 is the band of the probe TSDz structure. Therefore, the results of the gel experiment can prove the successful formation of the multifunctional triangular DNA biosensor. Since the fluorophore FAM is modified on the DNAzyme, the amount of DNAzyme used will affect the detection performance. Therefore, we further optimized the amount of DNAzyme used through gel electrophoresis and fluorescence experiments. Figure 2 As shown in B, lane 1 is the SS-L / Apt-DNA double-stranded band, and lanes 2-5 have fewer SS-L / Apt-DNA double-stranded bands as the amount of DNAzyme increases. When DNAzyme:SS-L:Apt-DNA=1.5:2:2, there is almost no SS-L / Apt-DNA double-stranded band remaining. Figure 2 As shown in C and 2D, as the amount of DNAzyme decreases, the fluorescence background signal becomes lower. When DNAzyme:SS-L:Apt-DNA=1:2:2, the fluorescence background signal can be ignored. Therefore, DNAzyme:SS-L:Apt-DNA=1:2:2 was finally selected for subsequent experiments. In addition, the synthesis time of the probe TSDz was also optimized, as shown in Figure 3 As shown in A and 3B, after 10 min of incubation, the fluorescence signal dropped to the lowest value.

[0033] We also analyzed the feasibility of the triangular DNA biosensor's response to target substances by fluorescence. Figure 3 As shown in C, when only GSH was added, the fluorescence increased only slightly, while when only the target Egr-1 was added, the fluorescence rebounded to a higher level. When GSH and Egr-1 were added at the same time, the fluorescence rebounded with a higher intensity, indicating that the strategy of using the triangular DNA biosensor's glutathione (GSH) response to detect Egr-1 is feasible.

[0034] Example 3: Application of the triangular DNA biosensor.

[0035] (1) Response of the comparative probe (TDz) of the triangular DNA biosensor to different concentrations of Egr-1 and its kinetic analysis

[0036] A comparative probe (TDz) for triangular DNA biosensor was designed. The probe TDz does not contain disulfide bonds and cannot respond to GSH. Different concentrations of Egr-1 were added to the probe (100 nM) for evaluation to determine the sensitivity of the probe. Fluorescence spectra ( Figure 4 A) shows that as the concentration of Egr-1 increases, the measured fluorescence signal also gradually increases. When the concentration of Egr-1 is 75 nM, the fluorescence ratio reaches a maximum value of 1.36 ( Figure 4 B), and the fluorescence intensity did not show a good linear relationship with the Egr-1 concentration, indicating that the probe without a designed disulfide bond was not sensitive to Egr-1. In addition, the probe was subjected to kinetic analysis ( Figure 4 C), as the incubation time after adding the target increases, F / F0 also increases, reaching the maximum value after 20 min, indicating that the probe design has a short response time to the target and a fast detection speed.

[0037] (2) Response of triangular DNA biosensor (TSDz) to different concentrations of Egr-1 and its kinetic analysis

[0038] The sensitivity of the probe was determined by adding different concentrations of Egr-1 (5 mM GSH) to 100 nM probe TSDz. Figure 5 As shown in A, as the concentration of Egr-1 increases, the measured fluorescence signal also gradually increases. In the range of 0-1nM, the fluorescence intensity has a good linear relationship with the concentration of the target ( Figure 5 B), its linear equation is Y = 14.55C + 47.40, R 2 =0.9991, where Y represents the fluorescence intensity and C is the concentration of Egr-1. The detection limit is 0.25 nM according to the 3σ / k theory. The results show that the triangular DNA biosensor (TSDz) has good detection effect, high sensitivity and low detection limit. The probe was also subjected to kinetic analysis. In the presence of different concentrations of Egr-1, the maximum fluorescence value was reached after 20 min ( Figure 5 C), indicating that the probe detects the target quickly.

[0039] (3) Selectivity experiment of triangular DNA biosensor (TSDz)

[0040] To investigate the selectivity of triangular DNA biosensor (TSDz) for Egr-1, we selected Egr-1 at a concentration (100 nM) and three other mRNAs (TK1, Survivin, and c-myc) for selectivity analysis experiments. Figure 6As shown in A, the fluorescence ratio F / F0 of the sensor is maximum only in the presence of Egr-1, indicating that the strategy is highly selective for the target (the above probe concentration is 100 nM).

[0041] (4) Comparative experiment of triangular DNA biosensor (TSDz) in PBS buffer and serum (1%)

[0042] The comparative experiment of the sensor in PBS buffer and serum (1%) can determine whether the sensor can be used for detection in actual samples. Figure 6 As shown in B, at different concentrations of Egr-1, the detection performance of the probe in serum is not much different from that in PBS, indicating that the sensor can well detect the target in the actual sample.

[0043] (5) DNase I and temporal stability of triangular DNA biosensor (TSDz)

[0044] The enzyme stability of the biosensor is crucial for accurate detection of the target. Therefore, we investigated the deoxyribonuclease I of the sensor. Figure 7 As shown in A, the probe TSDz can remain stable for 12 hours in the presence of DNaseⅠ (500U / L) without affecting its detection performance. The fluorescence of Egr-1 detected within 12 hours also did not change significantly. At the same time, we also explored the temporal stability of the probe. The probe maintained a very stable structure within 7 days ( Figure 7 B) The above results show that the DNase I and time stability of the triangular DNA biosensor of the present invention are very satisfactory.

Claims

1. Construction and application of a multifunctional triangular DNA biosensor, which is characterized by the following synthesis steps: DNAzyme (10 μL, 20 μM), SS-L and Apt-DNA (20 μL, 20 μM) are mixed evenly with 150 μL PBS buffer and incubated at 37°C for 20 min in one pot to obtain a multifunctional triangular DNA probe (named TSDz).

2. The construction and application of a multifunctional triangular DNA biosensor according to claim 1, wherein: (1) The multifunctional triangular DNA biosensor consists of three functional DNA single strands, namely DNAzyme, SS-L, and Apt-DNA; (2) In the absence of GSH and the target Egr-1 mRNA, the FAM modified on the 5' end of the DNAzyme and the BHQ1 on the 3' end of the Apt-DNA are close to each other, resulting in the quenching of the FAM fluorescence by BHQ1, resulting in a lower background signal; (3) When only GSH is present, the disulfide bonds in SS-L are reduced and broken by GSH, but without the target Egr-1 mRNA, the DNAzyme on TSDz will not bind to the target to produce fluorescence; (4) When only the target Egr-1 mRNA is present but GSH is absent, Egr-1 mRNA will bind to the 5' end of TSDz, resulting in a small increase in fluorescence; (5) When GSH and the target Egr-1 mRNA are present at the same time, GSH cuts the disulfide bonds to release some binding sites. Under the action of the target Egr-1 mRNA, the DNAzyme on TSDz can be replaced to produce a large amount of fluorescence. At the same time, under the action of magnesium ions, Egr-1 mRNA will be cut to achieve gene silencing.

3. The construction and application of a multifunctional triangular DNA biosensor according to claim 1, wherein the sequences of DNAzyme, SS-L and Apt-DNA are: DNAzyme: 5'-AACCGCGGCCAGGCTAGCTACAACGACCTGGACGA-3'; SS-L: 5'-TAGTGTATATGCGTTTTCGTC / iHS-SH / CAGGTCGTTGTTTCCGCGGTTTTTCGTATCTCAGAGGA-3'; Apt-DNA: 5'-ACGCATATACACTACGCAGTTGATCCTTTGGATACCCTG GTCCTCTGAGATACGA-3'.

4. The construction and application of a multifunctional triangular DNA biosensor according to claim 1 is characterized in that the biosensor detects Egr-1 mRNA and silences its expression through glutathione (GSH) response, and has high sensitivity and detection speed.

5. The construction and application of a multifunctional triangular DNA biosensor according to claim 1, wherein the fluorescence detection conditions are set as follows: Excitation wavelength: 488nm, excitation slit: 5nm, emission slit: 5nm, voltage: 800V.

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