Telomerase detection composition, kit and system based on DNA tetrahedral strand displacement reaction

Abstract

The invention discloses a telomerase detection composition, kit and system based on DNA tetrahedron strand displacement reaction, the composition comprises four single-chain sequence probes T1, T2, T3 and T4 used for assembling into a three-dimensional DNA tetrahedron, and the top of the three-dimensional DNA tetrahedron is provided with a suspension sequence P0; a probe P1 and a probe P2 each comprising a partial complementary sequence of P0; the 5'end of the probe P3 is marked with methylene blue; and a strand displacement reaction primer. The method has high sensitivity and good selectivity, and various cells can be distinguished through telomerase activity; meanwhile, the method also has the capability of screening a telomerase inhibitor; compared with a conventional detection scheme, the method does not need any other enzyme, only needs isothermal one-pot reaction, and can avoid complex operation and expensive reagents, so that the method has huge potential practical value for early diagnosis of telomerase-related diseases and drug research and development.

Description

Technical Field

[0001] The present invention relates to the technical field of biomacromolecule detection, and in particular to a telomerase detection composition, a kit and a system based on a DNA tetrahedral strand displacement reaction. Background Art

[0002] Telomere repeat amplification technology (TRAP) is currently the most commonly used telomerase detection and analysis technology. TRAP technology is based on the ability of telomerase to convert a telomere repeat sequence (TTAGGG) on its own template into n The telomere repeat fragment is synthesized to the 3' end of its substrate, and the extended telomere repeat fragment is amplified by PCR to determine the activity of telomerase. The general process includes: collecting cell samples and lysing them on ice, then separating the supernatant by freezing centrifugation, then adding templates, TRAP reaction buffer, dNTP Mix, primers and Taq DNA polymerase to the PCR reaction tube, and performing PCR amplification. Finally, the PCR product is subjected to electrophoresis and color development to analyze the activity of telomerase. However, the telomere repeat sequence amplification technology has the disadvantages of radioactive contamination, long detection time, difficulty in quantification, complex operation and high cost.

[0003] In addition, other technologies for detecting telomerase include new biosensor platforms based on fluorescence, SERS (surface enhanced Raman spectroscopy), photoelectrochemistry and other technologies. However, these methods require time-consuming operations, and the fluorescent substances used are unstable, and their background may interfere with quantitative analysis.

[0004] Therefore, it is necessary to improve the existing technology to provide a more reliable solution. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a telomerase detection composition, a kit and a system based on DNA tetrahedral strand displacement reaction in view of the deficiencies in the above-mentioned prior art.

[0006] The technical solution adopted by the present invention is: in the first aspect of the present invention, a telomerase detection composition based on DNA tetrahedron toehold-mediated strand displacement reaction is provided, comprising: Four single-stranded sequence probes T1, T2, T3, and T4 for assembling into a three-dimensional DNA tetrahedron, wherein the top of the three-dimensional DNA tetrahedron has an overhang sequence P0; Probes P1 and P2, both containing a partially complementary sequence to P0; Probe P3, whose sequence is completely complementary to a portion of the sequence of P0, and in addition, its 5' end is labeled with methylene blue; and a telomerase catalyzed reaction primer, which can be extended in the presence of telomerase, the extension product can hybridize with P0, so that probe P1 is displaced, probe P3 can simultaneously displace the extension product and probe P2, the released probes P1 and P2 are used again in the next round of displacement reaction, and assist in fixing probe P3 on the top of the three-dimensional DNA tetrahedron; When the composition is used for telomerase detection, the probes T1, T2, T3 and T4 are first assembled into a three-dimensional DNA tetrahedron and modified on an electrode, and then the modified electrode is incubated with the probes P1 and P2, and then the incubated electrode is immersed in a sample to be tested mixed with the probe P3 and primers for reaction, and finally the telomerase concentration in the sample to be tested is obtained by electrochemical detection of the electrode.

[0007] Preferably, the method for using the composition for telomerase detection is: first assemble probes T1, T2, T3, and T4 into a three-dimensional DNA tetrahedron and modify it on an electrode, then incubate the modified electrode with probes P1 and P2, and then immerse the incubated electrode in a sample to be tested mixed with probe P3 and primers for reaction, and finally measure the electrode by square wave voltammetry to obtain the SWV peak current, and then use a pre-constructed standard curve characterizing the relationship between the telomerase concentration and the SWV peak current to analyze and obtain the telomerase concentration in the sample to be tested.

[0008] Preferably, the sequences of T1, T2, T3, T4 and P0 are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively, and the 5' ends of the probes T2, T3 and T4 are all labeled with thiol.

[0009] Preferably, the sequences of P1, P2, P3 and the primer are shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively.

[0010] The second aspect of the present invention provides a use of the composition as described above in screening telomerase inhibitors.

[0011] The third aspect of the present invention provides a telomerase detection kit based on DNA tetrahedron toehold-mediated strand displacement reaction, which comprises the composition as described above.

[0012] In a fourth aspect, the present invention provides a telomerase detection system based on DNA tetrahedron toehold-mediated strand displacement reaction, which comprises the above-mentioned kit, modified electrode and electrochemical detection equipment.

[0013] Preferably, the method for detecting telomerase using the telomerase detection system based on DNA tetrahedral strand displacement reaction comprises the following steps: S1, electrode pretreatment; S2, assembling probes T1, T2, T3, and T4 into a three-dimensional DNA tetrahedron and modifying it on the pretreated electrode; S3, incubating the electrode modified with the three-dimensional DNA tetrahedron with the probes P1 and P2, and then immersing the incubated electrode in the sample to be tested mixed with the probe P3 and the primer for reaction; S4. After the reaction is completed, the electrode is taken out and measured by square wave voltammetry using an electrochemical detection device to obtain the SWV peak current. Finally, the telomerase concentration in the sample to be tested is obtained using a pre-constructed standard curve analysis that characterizes the relationship between the telomerase concentration and the SWV peak current.

[0014] Preferably, step S1 specifically comprises: The electrode was soaked in a piranha solution for 2-10 min, taken out and cleaned, and then polished into a mirror surface using P5000 sandpaper and alumina slurry in sequence, and then ultrasonically treated in ethanol and double distilled water for 2-10 min in sequence, and then rinsed with 0.2-1 M H2SO4 and scanned using cyclic voltammetry, and finally dried with nitrogen to obtain a pretreated electrode; the electrode was a gold electrode.

[0015] Preferably, step S2 specifically comprises: The probes T1, T2, T3, and T4, all with a concentration of 2-8 µM, were mixed in an equimolar ratio and added to a phosphate buffer containing NaCl. The mixture was incubated at 80-95 °C for 2-10 min. The product solution was cooled to room temperature. The pretreated electrode was inserted into the product solution and incubated for 4.5-18 h to obtain a three-dimensional DNA tetrahedron-modified electrode.

[0016] Preferably, step S3 specifically comprises: The electrode modified with three-dimensional DNA tetrahedron was incubated with probes P1 and P2 at concentrations of 0.4-1.6 µM for 0.5-2 h, and then the incubated electrode was rinsed and immersed in the test sample mixed with 0.25-1 µM probe P3 and 0.25-1 µM primers, and reacted at 37°C for 50-200 min.

[0017] The beneficial effects of the present invention are: The present invention provides a telomerase detection composition, a kit and a detection system based on a DNA tetrahedron (TDNA) strand displacement reaction, which can be used for accurate quantitative detection of telomerase activity. The present invention uses a constructed TDNA nanostructure to modify the electrode, providing an excellent molecular interaction interface; the present invention designs a toehold-mediated strand displacement amplification strategy initiated by a telomerase catalytic product, and the detection limit of the scheme is as low as 1 cell / mL; in addition to having high sensitivity, the method also has good selectivity and can distinguish a variety of cells by telomerase activity; at the same time, the present invention also has the ability to screen telomerase inhibitors; compared with conventional detection schemes, the present invention does not require any other enzymes, only an isothermal one-pot reaction is required, and complex operations and expensive reagents can be avoided. Therefore, the present invention has great potential practical value for early diagnosis and drug development of telomerase-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the detection principle of the telomerase detection composition based on DNA tetrahedral strand displacement reaction of the present invention; Figure 2 Polyacrylamide gel electrophoresis images: (a) P0, (b) P0 and P1, (c) P0, P1, P2, (d) primer, P0, P1, P2, P3, (e) telomerase, primer, P0, P1, P2, P3, (f) P1, (g) P2; Figure 3 Polyacrylamide gel electrophoresis images of TDNA assembly: (a) T2, (b) T2, T3, (c) T2, T3, T4, (d) T1, T2, T3, T4; Figure 4 is the AFM image of TDNA; Figure 5 Cyclic voltammograms of bare electrode, TDNA modified electrode with P1 and P2 chains, TDNA modified electrode + P1 + P2 + P3 chains after chain displacement reaction without and with telomerase; Figure 6 The AC impedance spectra of bare electrode, TDNA modified electrode with P1 and P2 chains, TDNA modified electrode + P1 + P2 + P3 chains after chain displacement reaction without telomerase and with telomerase; Figure 7 Square wave voltammograms of bare electrode, TDNA modified electrode with P1 and P2 chains, TDNA modified electrode + P1 + P2 + P3 chains after strand displacement reaction without and with telomerase, and square wave voltammograms of telomerase-triggered MB localization without and with strand displacement cycles; Figure 8The square wave voltammograms were obtained using a series of telomerase concentrations. Fig. 9 is the calibration curve of the relationship between SWV peak current and telomerase concentration; Fig.10 is the standard curve of the relationship between SWV peak current and telomerase logarithmic concentration; Fig.11 is the relationship between telomerase activity and MST-312 concentration; Fig.12 To validate the specificity of the telomerase assay; Fig.13 These are the experimental results of using telomerase to identify different cell lines. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0020] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0021] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially. Example 1

[0022] This embodiment provides a telomerase detection composition based on DNA tetrahedral strand displacement reaction, comprising: Four single-stranded sequence probes T1, T2, T3, and T4 used to assemble into a three-dimensional DNA tetrahedron, the top of which has an overhang sequence P0; Probes P1 and P2, both containing a partially complementary sequence to P0; probe P3, whose 5′ end was labeled with methylene blue (MB); and a primer, which can be extended in the presence of telomerase, the extension product can hybridize with P0, so that probe P1 is displaced, probe P3 can displace the extension product and probe P2, and the released probes P1 and P2 are used again in the next round of displacement reaction and assist in fixing probe P3 on the top of the three-dimensional DNA tetrahedron; The method for using the composition for telomerase detection is as follows: firstly assembling probes T1, T2, T3 and T4 into a three-dimensional DNA tetrahedron and modifying the probes on an electrode, then incubating the modified electrode with probes P1 and P2, then immersing the incubated electrode in a sample to be tested mixed with probe P3 and primers for reaction, finally measuring the electrode by square wave voltammetry to obtain SWV peak current, and then analyzing the telomerase concentration in the sample to be tested using a pre-constructed standard curve characterizing the relationship between the telomerase concentration and the SWV peak current.

[0023] In this embodiment, the sequences of the above compositions are shown in Table 1 below: Table 1

[0024] Among them, the underlined sequence is the complementary pairing sequence with P3.

[0025] This embodiment also provides a telomerase detection kit based on DNA tetrahedron toehold-mediated strand displacement reaction, which includes the above composition.

[0026] This embodiment also provides a telomerase detection system based on DNA tetrahedron toehold-mediated strand displacement reaction, which includes the above-mentioned kit, electrode and electrochemical detection equipment. The method for telomerase detection by the system includes the following steps: S1. Electrode pretreatment: The electrode was immersed in a piranha solution (98wt% H2SO4: 30wt% H2O2 volume ratio = 3:1) for 5 min, taken out and cleaned, and then polished into a mirror surface using P5000 sandpaper and alumina slurry (three types of alumina particle sizes were used: 1, 0.3, and 0.05 µm). It was then ultrasonically treated in ethanol and double distilled water for 5 min, and then rinsed with 0.5 M H2SO4 and scanned using cyclic voltammetry (CV). After electrochemical cleaning, it was blown dry with nitrogen to obtain a pretreated electrode; the electrode was a gold electrode.

[0027] S2. Assemble probes T1, T2, T3, and T4 into a three-dimensional DNA tetrahedron and modify it on the pretreated electrode: Probes T1, T2, T3, and T4, all with a concentration of 4 µM, were mixed in an equimolar ratio and added to 10 mM phosphate buffer (PBS) containing 0.1 M NaCl (pH 7.4). The mixture was incubated at 95 °C for 5 min. The product solution was cooled to room temperature, and the pretreated electrode was inserted into the product solution and incubated for 9 h to obtain a three-dimensional DNA tetrahedron-modified electrode.

[0028] S3. Incubate the electrode modified with three-dimensional DNA tetrahedron with probes P1 and P2 at a concentration of 0.8 µM for 1 h, then rinse the incubated electrode and immerse it in the test sample mixed with 0.5 µM probe P3 and 0.5 µM primer, and react at 37 °C for 100 min.

[0029] S4. After the reaction is completed, the electrode is taken out and measured by square wave voltammetry using an electrochemical detection device to obtain the SWV peak current. Finally, the telomerase concentration in the sample to be tested is obtained using a pre-constructed standard curve analysis that characterizes the relationship between the telomerase concentration and the SWV peak current.

[0030] Detection principle: Reference Figure 1 In the detection system provided in this embodiment, a three-dimensional DNA tetrahedron (TDNA) is assembled from four single-stranded DNAs (T1, T2, T3, and T4), and then modified on the surface of a gold electrode; the three vertices of the TDNA are labeled with thiols, which facilitates firm fixation through gold-sulfur bonds, and the remaining linear chain, called P0, serves as a template for a chain displacement reaction, which first independently positions P1 and P2 through a partial hybridization reaction. In the presence of telomerase, the primer sequence can be extended, and the product can hybridize with P0 through the foothold of P0 / P1 / P2, and the P1 chain is displaced to form another foothold; after further introduction of P3, the extended product and P2 can be displaced, and the released chain can be used again for the next round of displacement reaction and assist in fixing multiple P3 chains on the top of the TDNA. Since P3 is labeled with methylene blue (MB), the initial concentration of telomerase can be evaluated by recording and analyzing the electrochemical reaction. Due to the optimized design of the DNA nanostructure, the entire process does not involve the use of additional enzymes, and the isothermal one-pot reaction system is very efficient.

[0031] In this embodiment, pre-prepared telomerase solutions of different concentrations are used as test samples for relevant detection, and the preparation method is as follows: (1) Prepare DMEM medium containing 10wt% fetal bovine serum, add 1w% penicillin / streptomycin mixture, culture HeLa cells at 37℃ and 5% carbon dioxide, wash with PBS, treat with trypsin and collect. Wash the cells further and resuspend them in 10 mM Tris-HCl buffer (pH 7.4), which contains 1 mM MgCl2, 1 mM EGT A, 0.1 mM PMSF, 0.5% CHAPS (mass volume ratio, w / v) and 10% glycerol (volume ratio, v / v). After placing on ice for 30 minutes, centrifuge at 4℃ (12000 rpm) for 30 minutes. Repeat 2-3 times and collect the supernatant in a new pyrogen-free and RNase-free tube for telomerase extraction.

[0032] (2) After telomerase was extracted, it was diluted to the following concentration series using telomerase buffer (containing: 20 mM Tris-HCl, 1 mM Egta, 63 mM KCl, 1.5 mM MgCl2, 10 mM dNTP, 0.05% Tween 20, pH 8.3): 10, 50, 100, 500, 1000, 5000, 10000, 50000, 100000, 500000 cells / mL for subsequent detection.

[0033] The electrochemical detection device in this embodiment is a CHI660D electrochemical analyzer (CH Instruments, China), which adopts a traditional three-electrode system, including a saturated calomel reference electrode, a platinum wire auxiliary electrode and the modified three-dimensional DNA tetrahedron prepared above.

[0034] In this example, 5 mM [Fe(CN)6] 3- / 4- Electrolyte solutions for cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements. Prepare 20 mM Tris−HCl (pH 7.4) containing 140 mM NaCl and 5 mM MgCl2 as the electrolyte for square wave voltammetry (SWV) measurements. Prepare 50 μM [Ru(NH3)6] 3+ A 10 mM Tris−HCl (pH 7.4) solution was used for chronocoulometry (CC) measurements.

[0035] In this embodiment, the electrochemical detection parameters are: bias potential 0.21 V, amplitude 5 mV, frequency range 0.1 ~100000 Hz (EIS); scan range 0.6 ~ -0.1 V, scan rate 0.1 V / s (for CV); modulation amplitude 25 mV, potential step 4 mV, frequency 70 Hz (for SWV). Performance Characterization and Testing

[0036] 1. Characterization of DNA Tetrahedron The TDNA assembly products were identified by non-denaturing polyacrylamide gel electrophoresis (PAGE) and atomic force microscopy (AFM). First, the DNA hybridization products were injected into the prepared gel and electrophoresed in 90 mM tris-borate buffer (1 mM EDTA, pH 8.0) with a voltage of 120 V and a time of 50 min. The obtained gel was stained with 4S Red Plus and photographed under ultraviolet light using a gel DocTM XR+ imaging system (Bio-Rad, USA). AFM imaging was achieved using an Agilent5500 atomic force microscope (Agilent, USA). 10 μL of DNA hybridization products were deposited on a mica sheet, washed three times with double-distilled deionized water after 7 min, dried with nitrogen, and the images were taken in the tapping mode with the parameters of a tip radius of 2 nm, a tip spring constant of 40 N / m, and a resonance frequency of 325 kHz.

[0037] like Figure 2 The following are polyacrylamide gel electrophoresis images, where: (a) P0, (b) P0 and P1, (c) P0, P1, P2, (d) primer, P0, P1, P2, P3, (e) telomerase, primer, P0, P1, P2, P3, (f) P1, (g) P2. Lane a shows the band of P0, and lane b shows the expected double-stranded P0 / P1 with a larger molecular weight. In addition, the band in lane c belongs to P0 / P1 / P2, and the electrophoresis speed is slightly slower. After the primer is mixed with P3, the band remains unchanged, indicating that the double-stranded cannot directly interact with the primer and P3. Telomerase can extend the primer, triggering the strand displacement reaction to generate P0 / P3 products. It is faster than P0 / P1 / P2. In addition, the bands of released P1 and P2 can also be observed (lane e). The position of the bands shows that the strand displacement reaction is highly dependent on the target telomerase.

[0038] The assembly of TDNA (called tetrahedron in the figure) was also characterized by PAGE, e.g. Figure 3 As shown, among them: (a) T2, (b) T2, T3, (c) T2, T3, T4, (d) T1, T2, T3, T4; the band in lane a is single-stranded T2, and the band in lane b is a double-stranded formed by T2 and T3, with a higher molecular weight. Similarly, the bands in lanes c and d are DNA nanostructures formed by 3 and 4 chains, respectively. The formed TDNA has the largest molecular weight, indicating the successful assembly of the three-dimensional DNA nanostructure.

[0039] The formed TDNA structure was imaged by AFM technique, e.g. Figure 4 As shown, these bright spots belong to the formed DNA nanostructures.

[0040] 2. Electrochemical characterization In this example, different electrochemical phenomena of the TDNA interface before and after the reaction were studied using CV, EIS and SWV. Figure 5 Cyclic voltammograms of bare electrode, tetrahedron-modified electrode with P1 and P2 chains (tetrahedron + P1 / 2), tetrahedron-modified electrode + P1 + P2 + P3 chains after chain displacement reaction without telomerase (tetrahedron + P1 / 2 + P3) and with telomerase (tetrahedron + P1 / 2 + telomerase + P3). A pair of clear current peaks were observed on the bare electrode. After TDNA modification and further P1 / P2 hybridization, the peak intensity decreased significantly. At the same time, the distance between the two peaks increased. The reason is that the immobilized DNA layer is negatively charged, which repels [Fe(CN)6] 3- / 4- , resulting in charge transfer resistance. After further incubation with P3, the curve hardly changed, indicating that P3 could not directly hybridize with P0 due to the retardation of P1 and P2. However, in the presence of telomerase and primer, P1 can be replaced and create a new foothold. Therefore, P3 participates in the second strand displacement reaction, displacing the extended primer and P2 sequence. Compared with the branched P0 / P1 / P2, the complete double-stranded chain (P0 / P3) has less steric hindrance. Therefore, the intensity of the CV peak current is restored to a certain extent, and the distance between the two peaks is reduced. Similar conclusions were obtained through EIS characterization. A typical AC impedance spectrum usually contains a low-frequency linear part related to mass transfer and a high-frequency semicircular part related to electron transfer. The diameter of the semicircle represents the interfacial charge transfer resistance ( R ct ).

[0041] like Figure 6 Shown are the AC impedance spectra of the bare electrode, tetrahedron-modified electrode with P1 and P2 chains (tetrahedron + P1 / 2), tetrahedron-modified electrode + P1 + P2 + P3 chains after chain displacement reactions without telomerase (tetrahedron + P1 / 2 + P3) and with telomerase (tetrahedron + P1 / 2 + telomerase + P3); the bare electrode is a straight line, and the TDNA / P1 / P2 modified electrode is a large semicircle. The diameters were basically the same after incubation with P3, indicating that the complementary sequence was hidden. However, with the extension of telomerase and the toehold-mediated chain displacement cycle, the diameter became significantly smaller, verifying the reduction of branch chains by releasing them. R ct , these trends are consistent with the CV results.

[0042] Since P3 is marked as MB, SWV is used to detect MB signals. Figure 7Shown are square wave voltammograms of bare electrode, tetrahedron-modified electrode with P1 and P2 chains (tetrahedron + P1 / 2), tetrahedron-modified electrode + P1 + P2 + P3 chains after strand displacement reaction without telomerase (tetrahedron + P1 / 2 + P3) and with telomerase (tetrahedron + P1 / 2 + telomerase + P3), as well as square wave voltammograms of telomerase-triggered MB localization without and with strand displacement cycles. Only in the presence of telomerase can P3 be localized and form a distinct current peak. By analyzing the SWV peak intensity, the number of MB molecules enriched on TDNA can be assessed. We further introduced direct measurement with primers labeled with MB instead of P3 with strand displacement cycles. In the presence of telomerase, primer MB can also be extended and then hybridized with P0 on the top of TDNA. However, without strand displacement cycles, local MB molecules are limited and the peak intensity is much lower than the case of P3-initiated amplification, proving the necessity of cycle design.

[0043] 3. Standard curve construction The system in Example 1 was used to detect a series of concentrations of standard telomerase prepared above, such as Figure 8 As shown, with increasing telomerase concentration, the SWV peak current increased as expected.

[0044] The relationship between SWV peak current and telomerase concentration is as follows Fig. 9 As shown, its linear range is 10 - 10 4 Cells / mL( Fig.10 ), the equation is as follows: y = 1.98 + 1.22 x ( R 2 = 0.99; n = 4, n represents 4 repeated experiments), where y is the recorded SWV peak current, x is the logarithmic concentration of telomerase. The detection limit was calculated to be 1 cell / mL (S / N = 3).

[0045] 4. Evaluation of the effect of telomerase inhibitors Inhibition of telomerase is a promising approach to inhibit uncontrolled cell proliferation. In order to verify the performance of the biosensor as an inhibitor evaluation platform, MST-312 was used as a model inhibitor in this example.

[0046] MST-312 is a chemically modified derivative of green tea epigallocatechin gallate (EGCG) and a telomerase inhibitor. The MST-312 solid was first dissolved in a certain amount of DMSO and then dissolved in water to ensure that the final concentration of DMSO was one thousandth. Different masses of MST-312 were weighed and prepared into standard solutions with final concentrations of 0.1 µM, 0.3 µM, 0.5 µM, 0.7µM, 0.9 µM, 1 µM, 2 µM, 5 µM, 7 µM, and 10 µM, respectively, and mixed with telomerase.

[0047] The TDNA modified electrode was incubated with P1 and P2 (0.8 µM) for 1 h. After rinsing, the electrode was immersed in a mixture of primers, P3 (0.5 µM) and telomerase containing different concentrations of MST-312 at 37°C. After 100 min of reaction, the electrode was rinsed and measured by square wave voltammetry (SWV). Since MST-312 has an inhibitory effect on telomerase, the amplification effect of telomerase on primers decreases with the increase of MST-312 concentration, further inhibiting the downstream SDA reaction and causing a decrease in electrical signal output. As MST-312 increases, telomerase activity gradually decreases (e.g. Fig.11 ), and decreased in a concentration-dependent manner, IC 50 The concentration was about 0.7 μM, and the test results were accurate and consistent with the drug properties of MST-312. Based on this, the telomerase detection method can be used for the screening and effect evaluation of different types of telomerase inhibitors, and the reaction process is simple and easy to operate.

[0048] 5. Specificity verification of telomerase detection Other protein solutions were used to verify the specific selectivity of the telomerase detection method. Interfering proteins included BSA (bovine serum albumin), VEGF (vascular epidermal growth factor), trypsin, EcoRI, DpnI and Nb.BbvCI, and a solution without BSA was used as a blank control. The TDNA modified electrode was incubated with P1 and P2 (0.8 µM) for 1 h. After rinsing, the electrode was immersed in different protein solutions mixed with primers and P3 (0.5 µM) at 37°C. After 100 min of reaction, the electrode was rinsed and measured by square wave voltammetry (SWV). Since these proteins cannot extend the primer sequence and trigger SDA to form a signal amplification effect, compared with interfering proteins, only telomerase will produce a stronger SWV peak current when detected using this method. The other experimental groups are almost indistinguishable from the blank control, and the peak current is very weak ( Fig.12). This indicates that the above interfering proteins cannot trigger a reaction in SWV to output a higher MB signal, which is the same as the case in the control experiment, because they cannot extend the primer sequence and trigger the following SDA. On the contrary, a significant peak current was generated in the presence of telomerase, indicating that the detection method in this study is highly selective for the target of telomerase and is not easily interfered by other proteins.

[0049] 6. Telomerase is used to identify different cell lines A portion of the collected HeLa cells was taken out and inactivated by heating at 80°C for 10 min. RPMI-1640 containing 10% fetal bovine serum was prepared, and a 1% penicillin / streptomycin mixture was added. L02 cells were cultured at 37°C and 5% carbon dioxide. DMEM medium containing 10% fetal bovine serum was prepared, and a 1% penicillin / streptomycin mixture was added. MCF-7 and HepG2 cells were cultured at 37°C and 5% carbon dioxide. Telomerase was extracted from HeLa, L02, MCF-7, HepG2 and heat-inactivated HeLa cells (all at a concentration of 10 4 cells / mL).

[0050] The TDNA modified electrode was incubated with P1 and P2 (0.8 µM) for 1 h, and then rinsed and immersed in a mixed solution of primers, P3 (0.5 µM) and telomerase extracted from different cell lines at 37 °C. After 100 min of reaction, the electrode was rinsed and measured by square wave voltammetry (SWV). Fig.13 As shown in the figure, telomerase extracted from HeLa cells produced a high peak current. However, heat treatment can inactivate telomerase activity (Heated HeLa), and its electrochemical reaction is similar to that of the control group. In addition, SWV peaks were also observed in L02, MCF-7 and HepG2 cells. Different values ​​represent different telomerase expression levels. Telomerase extracted from different cell lines produced different degrees of peak current, indicating that this telomerase detection method can be extended to the detection of telomerase in different cell lines.

[0051] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A telomerase detection composition based on DNA tetrahedral strand displacement reaction, characterized in that: include: Four single-stranded sequence probes T1, T2, T3, and T4 for assembling into a three-dimensional DNA tetrahedron, wherein the top of the three-dimensional DNA tetrahedron has an overhang sequence P0; Probes P1 and P2, both containing a partially complementary sequence to P0; Probe P3, whose sequence is completely complementary to a portion of the sequence of P0, and in addition, its 5' end is labeled with methylene blue; and a telomerase catalyzed reaction primer, which can be extended in the presence of telomerase, the extension product can hybridize with P0, so that probe P1 is replaced, probe P3 can simultaneously replace the extension product and probe P2, the released probes P1 and P2 are used again in the next round of replacement reaction, and assist in fixing probe P3 on the top of the three-dimensional DNA tetrahedron; When the composition is used for telomerase detection, the probes T1, T2, T3 and T4 are first assembled into a three-dimensional DNA tetrahedron and modified on an electrode, and then the modified electrode is incubated with the probes P1 and P2, and then the incubated electrode is immersed in a sample to be tested mixed with the probe P3 and primers for reaction, and finally the telomerase concentration in the sample to be tested is obtained by electrochemical detection of the electrode.

2. The telomerase detection composition based on DNA tetrahedral strand displacement reaction according to claim 1, characterized in that: The method for using the composition for telomerase detection is as follows: firstly assembling probes T1, T2, T3 and T4 into a three-dimensional DNA tetrahedron and modifying the probes on an electrode, then incubating the modified electrode with probes P1 and P2, then immersing the incubated electrode in a sample to be tested mixed with probe P3 and primers for reaction, finally measuring the electrode by square wave voltammetry to obtain SWV peak current, and then analyzing the telomerase concentration in the sample to be tested using a pre-constructed standard curve characterizing the relationship between the telomerase concentration and the SWV peak current.

3. The telomerase detection composition based on DNA tetrahedral strand displacement reaction according to claim 2, characterized in that: The sequences of T1, T2, T3, T4 and P0 are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively, and the 5' ends of the probes T2, T3 and T4 are all labeled with thiol; The sequences of P1, P2, P3 and primers are shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively.

4. Use of the composition according to any one of claims 1 to 3 in screening telomerase inhibitors.

5. A telomerase detection kit based on DNA tetrahedral strand displacement reaction, characterized in that: It comprises the composition according to any one of claims 1 to 3.

6. A telomerase detection system based on DNA tetrahedral strand displacement reaction, characterized in that: It comprises the reagent kit as claimed in claim 5, an electrode and an electrochemical detection device.

7. The telomerase detection system based on DNA tetrahedral strand displacement reaction according to claim 6, characterized in that: The method for performing telomerase detection in the system comprises the following steps: S1, electrode pretreatment; S2, assembling probes T1, T2, T3, and T4 into a three-dimensional DNA tetrahedron and modifying it on the pretreated electrode; S3, incubating the electrode modified with the three-dimensional DNA tetrahedron with the probes P1 and P2, and then immersing the incubated electrode in a sample to be tested mixed with the probe P3 and primers for reaction; S4. After the reaction is completed, the electrode is taken out and measured by square wave voltammetry using an electrochemical detection device to obtain the SWV peak current. Finally, the telomerase concentration in the sample to be tested is obtained using a pre-constructed standard curve analysis that characterizes the relationship between the telomerase concentration and the SWV peak current.

8. The telomerase detection system based on DNA tetrahedral strand displacement reaction according to claim 7, characterized in that: Step S1 is specifically as follows: The electrode was soaked in a piranha solution for 2-10 min, taken out and cleaned, and then polished into a mirror surface using P5000 sandpaper and alumina slurry in sequence, and then ultrasonically treated in ethanol and double distilled water for 2-10 min in sequence, and then rinsed with 0.2-1 M H2SO4 and scanned using cyclic voltammetry, and finally dried with nitrogen to obtain a pretreated electrode; the electrode was a gold electrode.

9. The telomerase detection system based on DNA tetrahedral strand displacement reaction according to claim 7, characterized in that: Step S2 is specifically as follows: The probes T1, T2, T3, and T4, all with a concentration of 2-8 µM, were mixed in an equimolar ratio and added to a phosphate buffer containing NaCl. The mixture was incubated at 80-95 °C for 2-10 min. The product solution was cooled to room temperature. The pretreated electrode was inserted into the product solution and incubated for 4.5-18 h to obtain a three-dimensional DNA tetrahedron-modified electrode.

10. The telomerase detection system based on DNA tetrahedral strand displacement reaction according to claim 9, characterized in that: Step S3 is specifically as follows: The electrode modified with three-dimensional DNA tetrahedron was incubated with probes P1 and P2 at concentrations of 0.4-1.6 µM for 0.5-2 h, and then the incubated electrode was rinsed and immersed in the test sample mixed with 0.25-1 µM probe P3 and 0.25-1 µM primers, and reacted at 37 °C for 50-200 min.

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