A method for detecting telomerase activity and a detection kit

Abstract

The application discloses a telomerase activity detection method and a detection kit. The method comprises the following steps: providing a functional DNA module, wherein the functional DNA module comprises a telomerase primer and a blocked split DNAzyme, the blocked split DNAzyme comprises a DNAzyme nucleic acid chain and at least two blocking chains, and the blocking chains are hybridized with the DNAzyme nucleic acid chain; mixing a sample to be detected with the functional DNA module, wherein a telomerase recognizes and extends the telomerase primer to generate an extension product; the extension product replaces the blocking chains from the blocked split DNAzyme through a strand displacement reaction, so that the DNAzyme nucleic acid chain restores catalytic activity; the DNAzyme nucleic acid chain cuts a substrate chain and generates a signal; and the signal intensity is detected to determine telomerase activity. Through the blocked structure of the blocked split DNAzyme and the blocking chains and by using a telomerase extension reaction and a strand displacement reaction, the application realizes high-sensitivity and low-background detection of telomerase activity.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method and kit for detecting telomerase activity. Background Technology

[0002] Telomeres are DNA-protein complexes containing telomeric DNA repeat sequences (TTAGGG) at the ends of linear chromosomes in eukaryotes. They are considered "terminal caps" protecting the integrity of the genome, and their length gradually shortens during DNA replication in each cell cycle. When telomeres shorten to a critical length, programmed cell death is triggered, leading to replicative senescence or apoptosis. Telomerase is a large ribonucleoprotein complex responsible for the stepwise synthesis of telomeric DNA repeat sequences (TTAGGG) at the 3′ ends of linear chromosomes, thereby reversing DNA loss during each replication cycle and playing a crucial role in maintaining telomere length. In 1985, Greider and Blackburn first discovered a reverse transcriptase-active enzyme in Tetrahymena cells that could synthesize telomeric repeat sequences using its own RNA as a template, and named it telomerase. It was subsequently found in other ciliates. Further research revealed that telomerase activity was detected in human HeLa cells and discovered in various human cancers. Since then, telomerase research has expanded from the ciliate field to the general field of eukaryotic biology.

[0003] Telomerase consists of two core components: one is telomerase reverse transcriptase (TERT), encoded by the human hTERT gene located in the 5p15.33 region of chromosome 5, which serves as the catalytic protein subunit; the other is human telomerase RNA (hTR) or human telomerase RNA fraction (hTERC), encoded by the hTERC gene located in the 3q26 region of chromosome 3, which is an essential RNA component. hTR not only serves as a template in telomere DNA synthesis but also participates in telomerase-catalyzed reactions, localization, and assembly processes.

[0004] Studies have shown that telomerase activity is typically low or undetectable in normal somatic cells, but it is frequently reactivated in cancer cells, leading to telomere elongation at the ends of chromosomes and exhibiting high expression levels. When telomerase activity is sufficient, stem cells, germ cells, and lymphocytes can sustainably maintain telomere length, ensuring genomic stability and long-term proliferative potential. However, in individuals with telomerase deficiency or insufficient activity, progressive telomere shortening triggers DNA damage responses, leading to "telomere shortening syndrome" manifestations such as bone marrow failure, pulmonary fibrosis, and cirrhosis. Furthermore, telomeres and telomerase play crucial roles in various diseases, such as cirrhosis and liver cancer, prostate cancer, and endometrial lesions. Research results show that approximately 90% of malignant tumors directly upregulate TERT transcription through TERT promoter mutations, super-enhancer reprogramming, or viral oncoproteins (such as HPV-E6), causing continuous telomere elongation in cancer cells and evading immune clearance, leading to cancer development and progression. This makes telomerase a universal biomarker and potential target for tumor diagnosis, prognostic assessment, and treatment. Telomerase's abnormal expression and tissue specificity in major diseases such as tumors have made it an important biomarker for cancer detection. Therefore, developing highly sensitive and specific telomerase detection technologies has significant clinical value for early cancer diagnosis, prognostic assessment, and treatment monitoring.

[0005] Currently common telomerase detection techniques include Western blot (WB), telomere repeat sequence amplification (TRAP), real-time fluorescence TRAP (qTRAP), and TRAP-ELISA. While these techniques have made significant progress in telomerase activity analysis and clinical diagnosis, they still have unavoidable limitations, such as cumbersome operation, long processing time, the need for precise temperature control, high cost due to protease involvement, susceptibility to false negatives and false positives, and the presence of nonspecific interference. In recent years, isothermal enzyme-free signal amplification techniques have been introduced into telomerase detection, which can convert small amounts of telomerase extension products into a large amount of measurable signal at a constant temperature, showing significant research potential. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a method and kit for detecting telomerase activity. This invention achieves high-sensitivity, low-background detection of telomerase activity under isothermal, enzyme-free conditions by blocking split-type DNAzymes with a blocking structure consisting of at least two blocking strands and coupling telomerase extension and strand displacement reactions.

[0007] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution: This invention provides a method for detecting telomerase activity, comprising the following steps: A functional DNA module is provided, the functional DNA module comprising a telomerase primer and a blocking splitting DNAzyme, the telomerase primer being linked to the blocking splitting DNAzyme, the blocking splitting DNAzyme comprising a DNAzyme nucleic acid strand and at least two blocking strands, the DNAzyme nucleic acid strands assembling to form a splitting DNAzyme catalytic core, the at least two blocking strands hybridizing with the DNAzyme nucleic acid strand respectively to inhibit the catalytic activity of the DNAzyme nucleic acid strand; The sample to be tested is mixed with the functional DNA module, so that the telomerase in the sample to be tested recognizes and extends the telomerase primer, generating an extension product containing telomere repeat sequences. The extended product replaces the closed strand from the closed split DNAzyme through a chain substitution reaction, thereby restoring the catalytic activity of the DNAzyme nucleic acid chain; The DNAzyme nucleic acid strand cleaves the substrate strand and generates a detectable signal; The detectable signal is detected, and the activity of telomerase in the sample to be tested is determined based on the intensity of the detectable signal.

[0008] Furthermore, the DNAzyme nucleic acid chain is a splitting DNAzyme nucleic acid chain based on an 8-17 DNAzyme catalytic core, and the DNAzyme nucleic acid chain recognizes the rAG site in the substrate chain and cleaves the substrate chain at that site.

[0009] Furthermore, the reaction in which the DNAzyme nucleic acid chain cleaves the substrate chain is carried out in the presence of divalent metal ions; the divalent metal ions are magnesium ions.

[0010] Furthermore, the substrate chain is modified with a fluorescent group and a fluorescence quenching group at both ends, respectively, and the substrate chain generates a fluorescence signal after being cleaved; the fluorescent group is FAM, and the fluorescence quenching group is BHQ1.

[0011] Furthermore, after the DNAzyme nucleic acid strand cleaves the substrate strand, it detaches from the substrate strand and continues to recognize and cleave new substrate strands, generating signals in a cyclical manner.

[0012] Furthermore, the blocking split DNAzyme comprises a DNAzyme nucleic acid chain and two blocking strands. The DNAzyme nucleic acid chain includes three nucleic acid chains. The two blocking strands hybridize and assemble with the three nucleic acid chains to achieve double blocking of the blocking split DNAzyme.

[0013] Furthermore, the sample to be tested is a cell lysate, tissue sample, blood sample, or clinical biopsy sample.

[0014] Furthermore, determining the telomerase activity in the sample based on the intensity of the detectable signal includes: comparing the intensity of the detectable signal with a pre-established standard curve to calculate the number of telomerase-positive cells in the sample; the standard curve has the number of telomerase-positive cells as the abscissa and the intensity of the detectable signal as the ordinate.

[0015] Another aspect of the present invention provides a telomerase activity detection kit, comprising: A functional DNA module comprising a telomerase primer and a blocking splitting DNAzyme, wherein the telomerase primer is attached to the blocking splitting DNAzyme, the blocking splitting DNAzyme comprises a DNAzyme nucleic acid strand and at least two blocking strands, the DNAzyme nucleic acid strands assembling to form a splitting DNAzyme catalytic core, and the at least two blocking strands hybridizing with the DNAzyme nucleic acid strands respectively to inhibit the catalytic activity of the DNAzyme nucleic acid strands; A substrate chain that can be cleaved by a catalytically active DNAzyme nucleic acid chain; Reaction buffer.

[0016] Furthermore, the detection kit is used for in vitro telomerase activity detection or for in situ imaging analysis of intracellular telomerase activity.

[0017] The beneficial effects of this invention are as follows: This invention combines telomerase extension reaction, strand displacement reaction, and a blocking split DNAzyme signal amplification strategy to construct an isothermal enzyme-free telomerase activity detection method, which has the following beneficial effects: (1) This invention does not require the participation of protease, thus avoiding the problems of high cost and false negative / false positive caused by protease in the traditional TRAP method. The catalytic activity of DNAzyme nucleic acid chain depends on divalent metal ions, and does not require precise temperature control equipment or large instruments or common heating devices. The reaction can be completed at room temperature or physiological temperature, which has the advantages of simplicity and economy, and is easy to operate, which is conducive to clinical promotion.

[0018] (2) This invention employs at least two closed strands to hybridize with the DNAzyme nucleic acid strand, thereby performing multiple blocking on the split DNAzyme catalytic core. Compared with a single closed strand, at least two closed strands synergistically lock the catalytic core, effectively suppressing non-specific leakage caused by the unwinding equilibrium of the closed strand, significantly reducing the blank background signal, and improving the detection signal-to-noise ratio.

[0019] (3) This invention uses telomerase as the sole trigger source to mediate the strand displacement reaction and simultaneously activate the blocked split DNAzyme, achieving exponential fluorescence signal amplification dependent on telomerase activity. This allows the sensitivity and specificity to be determined by telomerase itself, resulting in high specificity. The multiple blocking structure of the blocked split DNAzyme further reduces the possibility of non-specific activation, ensuring that the detection specificity is determined by telomerase itself, effectively avoiding interference from non-target substances in complex biological samples.

[0020] (4) The DNAzyme nucleic acid chain designed in this invention is assembled to form a split DNAzyme catalytic core, which has a higher cleavage efficiency than traditional single-stranded DNAzyme. The DNAzyme nucleic acid chain with restored catalytic activity can cyclically cleave substrate chains with fluorescent groups and quenching groups modified at both ends. Each cleavage cycle releases a fluorescent signal, realizing telomerase activity-dependent signal amplification and significantly improving detection sensitivity.

[0021] (5) This invention has been successfully applied to the detection of telomerase activity in actual cell samples, clinical tumor tissues and normal tissues. It has good diagnostic performance and reliability, which is conducive to further exploring the application value of telomerase in clinical diagnosis, prognosis assessment and treatment monitoring. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the telomerase detection strategy of the present invention.

[0023] Figure 2 The figure shows the results of the feasibility study of TSDSD hybridization, displaying PAGE electrophoresis bands for different nucleic acid chain combinations.

[0024] Figure 3 The figure shows the feasibility study results of DNAzyme nucleic acid chain recognition and cleavage performance and S6 shielding ability, illustrating the agarose gel electrophoresis bands of the Sub substrate chain with and without magnesium ions and with and without S6 chains.

[0025] Figure 4 The figure shows the results of the feasibility study on telomerase activity and its response to the system. (A) is a PAGE electrophoresis image, and (B) is an agarose gel electrophoresis image.

[0026] Figure 5 The fluorescence spectrum of telomerase response to the system shows a comparison of fluorescence signal intensity between the blank group, the heat-inactivated telomerase group, and the telomerase group.

[0027] Figure 6 Electrophoresis diagrams for metal ion selectivity studies show the cleavage effect of the system in the presence of different divalent metal ions.

[0028] Figure 7 To detect Mg in the system 2+The optimized concentration results are shown in the figure, illustrating the effect of Mg concentration on fluorescence intensity. 2+ Concentration change curve.

[0029] Figure 8 The figure shows the optimized detection time of the system, illustrating the curve of fluorescence intensity changing with reaction time.

[0030] Figure 9 The optimized results of the system detection temperature are shown in the figure, which illustrates the curve of fluorescence intensity changing with reaction temperature.

[0031] Figure 10 The optimized pH detection results for the system are shown in the figure, which illustrates the curve of fluorescence intensity changing with the pH value of the system.

[0032] Figure 11 The figure shows the optimization results of the primer concentration for the detection system, illustrating the curve of fluorescence intensity changing with primer concentration.

[0033] Figure 12 The figure shows the results of the specificity test of telomerase, illustrating the comparison of fluorescence signal intensity between telomerase and different endogenous proteases.

[0034] Figure 13 The standard curve of the detection system shows the linear relationship between fluorescence signal and the number of HeLa cells.

[0035] Figure 14 The figure shows the results of the investigation on the anti-interference ability of the detection system in complex biological matrices, and compares the fluorescence signal intensity of the system after adding different interfering substances.

[0036] Figure 15 The graph shows the detection results of actual cell samples. (A) is the standard curve of the telomerase ELISA kit, and (B) is a comparison graph of the detection results of telomerase by this method and the ELISA kit.

[0037] Figure 16 The graph shows the results of telomerase activity detection in tissues of clinical cervical cancer patients and healthy adults. (A, E) are bar charts of telomerase fluorescence and concentration obtained by this method (A) and ELISA (E), respectively; (B, F) are box plots of telomerase activity expression in two different populations obtained by this method (B) and ELISA (F), respectively; (C, G) are thermograms of fluorescence and concentration intensity obtained by this method (C) and ELISA (G), respectively; (D) is the ROC analysis graph of the diagnostic performance of this method and ELISA for cervical cancer. Data are expressed as mean ± standard deviation (n=3). Statistical significance was calculated using Student's t-test, *P<0.05; **P<0.01. Detailed Implementation

[0038] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] To overcome the limitations of conventional telomerase detection methods, such as cumbersome operation, high cost due to protease catalysis, low telomerase activity in biological samples, and insufficient detection sensitivity, this invention eliminates the need for protease catalysis. It utilizes the coupling of telomerase extension reaction and strand displacement reaction, combined with a isothermal signal amplification strategy of blocking split DNAzymes, to construct an efficient, sensitive, and low-cost telomerase activity detection method.

[0040] The detection principle of this invention is as follows: Figure 1 As shown. First, a functional DNA module (TSDSD), namely a telomerase-responsive, closed-chain DNAzyme, is constructed. This module consists of a telomerase primer (TP) and a closed-chain DNAzyme linked together. The closed-chain DNAzyme contains a DNAzyme nucleic acid chain and at least two closed strands. Telomerase recognizes the TP sequence and catalyzes its extension, generating an extension product containing telomere repeat sequences. The extension product releases the closed strands from the TSDSD through a toehold-mediated strand displacement reaction, restoring the catalytic activity of the DNAzyme nucleic acid chain. The DNAzyme nucleic acid chain with restored catalytic activity (i.e., the open-chain DNAzyme nucleic acid chain, open-DSD) is then subjected to a reaction with divalent metal ions (Mg²⁺). 2+ Under catalysis, the DNAzyme nucleic acid chain specifically recognizes and cleaves the Sub substrate chain modified with fluorescence quenching pairs (FAM / BHQ1) at both ends, releasing a fluorescent signal. After one round of cleavage, the DNAzyme nucleic acid chain further catalyzes the cleavage of new Sub substrate chains, thereby amplifying the signal. Telomerase activity can be detected by detecting the intensity of the fluorescent signal.

[0041] This invention provides a method for detecting telomerase activity, comprising the following steps: A functional DNA module is provided, the functional DNA module comprising a telomerase primer and a blocking splitting DNAzyme, the telomerase primer being linked to the blocking splitting DNAzyme, the blocking splitting DNAzyme comprising a DNAzyme nucleic acid strand and at least two blocking strands, the DNAzyme nucleic acid strands assembling to form a splitting DNAzyme catalytic core, the at least two blocking strands hybridizing with the DNAzyme nucleic acid strand respectively to inhibit the catalytic activity of the DNAzyme nucleic acid strand; The sample to be tested is mixed with the functional DNA module, so that the telomerase in the sample to be tested recognizes and extends the telomerase primer, generating an extension product containing telomere repeat sequences. The extended product replaces the closed strand from the closed split DNAzyme through a chain substitution reaction, thereby restoring the catalytic activity of the DNAzyme nucleic acid chain; The DNAzyme nucleic acid strand cleaves the substrate strand and generates a detectable signal; The detectable signal is detected, and the activity of telomerase in the sample to be tested is determined based on the intensity of the detectable signal.

[0042] Specifically, determining the telomerase activity in the sample based on the intensity of the detectable signal includes: comparing the intensity of the detectable signal with a pre-established standard curve to calculate the number of telomerase-positive cells in the sample; the standard curve has the number of telomerase-positive cells as the abscissa and the intensity of the detectable signal as the ordinate.

[0043] In one embodiment, the DNAzyme nucleic acid chain is a splitting DNAzyme nucleic acid chain based on an 8-17 DNAzyme catalytic core, wherein the DNAzyme nucleic acid chain recognizes the 5′-rAG-3′ site in the substrate chain and cleaves the substrate chain at that site.

[0044] Furthermore, the reaction in which the DNAzyme nucleic acid chain cleaves the substrate chain is carried out in the presence of divalent metal ions; the divalent metal ions are magnesium ions.

[0045] For example, the substrate chain is modified with a fluorescent group and a fluorescence quenching group at both ends, and the substrate chain generates a fluorescence signal after being cleaved; the fluorescent group is FAM and the fluorescence quenching group is BHQ1.

[0046] In this invention, the DNAzyme nucleic acid strand detaches from the substrate strand after cleaving it, and continues to recognize and cleave new substrate strands, generating signals in a cyclical manner.

[0047] In one embodiment, the blocking split DNAzyme comprises a DNAzyme nucleic acid chain and two blocking strands, wherein the DNAzyme nucleic acid chain comprises three nucleic acid chains, and the two blocking strands hybridize and assemble with the three nucleic acid chains to achieve double blocking of the blocking split DNAzyme.

[0048] For example, the sample to be tested is a cell lysate, tissue sample, blood sample, or clinical biopsy sample.

[0049] Another aspect of the present invention provides a telomerase activity detection kit, comprising: A functional DNA module comprising a telomerase primer and a blocking splitting DNAzyme, wherein the telomerase primer is attached to the blocking splitting DNAzyme, the blocking splitting DNAzyme comprises a DNAzyme nucleic acid strand and at least two blocking strands, the DNAzyme nucleic acid strands assembling to form a splitting DNAzyme catalytic core, and the at least two blocking strands hybridizing with the DNAzyme nucleic acid strands respectively to inhibit the catalytic activity of the DNAzyme nucleic acid strands; A substrate chain that can be cleaved by a catalytically active DNAzyme nucleic acid chain; Reaction buffer.

[0050] Specifically, the detection kit is used for in vitro telomerase activity detection or for in situ imaging analysis of intracellular telomerase activity.

[0051] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The nucleotide sequences used in the following embodiments are shown in Table 1, and are numbered SEQ ID NO.1 to SEQ ID NO.6 in the sequence listing.

[0052] Table 1

[0053] I. Feasibility Verification Before conducting actual sample testing, the hybridization assembly of TSDSD, the cleavage activity of the 8-17 DNAzyme, the shielding ability of the S6 blocking strand, and the telomerase response of the system were first verified.

[0054] (1) Characterization of SDSD hybridization TSDSD was characterized by non-denaturing polyacrylamide gel electrophoresis (PAGE). Figure 2 As shown, lanes L1-S6 (lane 1), L1-L2 (lane 4), L1-L2-S6 (lane 5), L1-L3-S6 (lane 6), L1-L2-L3 (lane 9 in yellow box), L3-L1-S6-L2-L1-S6 (lane 7), and L3-L1-S6-TP-L2-L1-S6-TP (lane 8) have good hybridization ability. Lane 8 indicates that the TSDSD element of this scheme was successfully constructed.

[0055] (2) The recognition and cleavage performance of the DNAzyme nucleic acid chain on the substrate chain Sub and the shielding ability of the S6 blocking strand on the DNAzyme in the DNAzyme nucleic acid chain. Verification was performed using agarose gel electrophoresis. Figure 3As shown, comparing lanes 4 and 5, it can be seen that the Sub band in lane 5 has basically disappeared, indicating that the open-DSD containing the DNase sequence formed by adding L1, L2, and L3 is in the presence of divalent metal ions Mg. 2+ In its presence, the S6 chain can specifically recognize and cleave the Sub substrate chain. Comparing lane 7 with lanes 6 and 8, it can be seen that when the S6 chain is present, it can effectively shield the DNAzyme in open-DSD, preventing the DNAzyme from cleaving the Sub substrate chain (lanes 6 and 7). However, when the S6 chain is absent from the system, the Sub substrate chain is completely cleaved and the band disappears (lane 8). This demonstrates that the S6 chain has a good shielding ability against DNAzyme in open-DSD.

[0056] (3) Telomerase response to the system The feasibility of using PAGE, agarose gel electrophoresis, and fluorescence signal detection techniques to investigate telomerase activity and its responsiveness to the system was verified. Figure 4 A. Lanes 4, 5, and 6 show that the hybridization bands formed in lanes 5 and 6, where telomerase extract was added, are higher and brighter than those in lane 4, indicating that telomerase is active and has formed hybrid double strands of S6-TP extensions with more complementary bases. Figure 4 B. Comparing lanes 3 and 5 and lanes 7 and 9 respectively, firstly, after adding telomerase, a light band with a blue box appears (lanes 5 and 9), while this band does not appear in lanes without telomerase (lanes 3 and 7). It is preliminarily speculated that this is a hybridization band of S6-TP extension. Secondly, the Substrate Chain (Sub) band in the red box of the lane with telomerase is lighter than that in the lane without telomerase, indicating that telomerase may have extended the Substrate Chain and hybridized with S6, causing S6 to detach from the TSDSD hybridization complex. Subsequently, the DNAzyme in the formed open-DSD recognizes and cuts the Substrate Chain, thus making the band lighter.

[0057] The responsiveness of telomerase to the system was further demonstrated by fluorescence signal detection results. For example... Figure 5 As shown, after adding telomerase extract (TE) to the system, the fluorescence signal value was significantly higher than that of the blank group (KB) without telomerase extract and the negative control group (H-TE) with heat-inactivated telomerase extract. Moreover, the fluorescence values ​​of the blank group and the negative control group were almost equal and close to zero. Therefore, the scheme is feasible.

[0058] II. Optimization of Detection Conditions After verifying the feasibility of the principle, the detection conditions were systematically optimized to obtain the best detection performance.

[0059] (1) Metal ion specific screening The DNAzyme nucleic acid chain in this embodiment is a splitting DNAzyme nucleic acid chain based on the 8-17 DNAzyme catalytic core. 8-17 DNAzyme is a divalent metal ion-dependent DNA nucleoside enzyme; different metal ions exhibit varying catalytic effects on substrate recognition and cleavage. Therefore, exploring the cleavage activity of the DNAzyme nucleic acid chain in this embodiment on the target substrate "rAG" site under different divalent metal ion catalysis is crucial. The reaction system first denatured a mixture of TP (0.1 μM), L1 (0.1 μM), L2 (0.05 μM), L3 (0.05 μM), S6 (0.1 μM), Tris-HCl (50 mM, pH=7.5), and NaCl (50 mM) at 95 °C for 6 min, then naturally cooled to room temperature, thereby self-assembling to form TSDSD. Subsequently, telomerase extract (TE, 10...) was added to the reaction solution. 4 cells), Sub-FQ (0.1 μM), and respectively placed in Cu 2+ Mn 2+ Zn 2+ Fe 2+ Mg 2+ and Pb 2+ A 40 μL system (with metal ion concentration of 1 mM) was subjected to extension and enzymatic digestion reactions at 37 °C for 4 h. Figure 6 As shown in B, Mn 2+ and Mg 2+ The generation of a strong fluorescence signal indicates that both can efficiently catalyze the recognition and cleavage of the Sub-FQ substrate chain by the split DNAzyme nucleic acid chain in this embodiment, based on Mg. 2+ Non-toxic to cells and widely distributed within cells, it can directly trigger DNAzyme enzymes to catalyze substrate cleavage within cells. Therefore, Mg was selected in this embodiment. 2+ It was used as the optimal catalytic metal ion in subsequent experiments.

[0060] (2) Optimization of magnesium ion concentration To ensure efficient cleavage of the split DNAzyme nucleic acid chain at the "rAG" site of the Sub-FQ substrate chain, the study explored the role of Mg. 2+ The concentration has a significant impact on the cleavage reaction. The reaction system was first denatured at 95°C for 6 min by a mixture of TP (0.1 μM), L1 (0.1 μM), L2 (0.05 μM), L3 (0.05 μM), S6 (0.1 μM), Tris-HCl (50 mM, pH=7.5), and NaCl (50 mM), followed by natural cooling to room temperature, thus allowing self-assembly to form TSDSD. Subsequently, telomerase extract (TE, 10...) was added to the reaction solution.4 Cells), Sub-FQ (0.1 μM), and were placed in containers containing different concentrations of Mg. 2+ A 40 μL system was subjected to extension and enzymatic digestion reactions at 37 °C for 4 h. (The rest of the text appears to be a fragment and requires further context for accurate translation.) Figure 7 As shown, the fluorescence value changes gradually between 0 and 1 mM when the magnesium ion concentration is high. After 1 mM, the fluorescence value increases significantly with the increase of magnesium ion concentration, and the fluorescence signal tends to stabilize at 10 mM. Therefore, 10 mM was selected as the optimal magnesium ion concentration for subsequent studies.

[0061] (3) Optimization of reaction time To ensure rapid and efficient telomerase extension and DNAzyme digestion, exploring the effect of reaction time is crucial. Similarly, the reaction system was first denatured at 95°C for 6 min by a mixture of TP (0.1 μM), L1 (0.1 μM), L2 (0.05 μM), L3 (0.05 μM), S6 (0.1 μM), Tris-HCl (50 mM, pH=7.5), and NaCl (50 mM), followed by natural cooling to room temperature, thus allowing self-assembly to form TSDSD. Then, telomerase extract (TE, 10...) was added to the reaction solution. 4 cells), Sub-FQ (0.1 μM) and Mg 2+ (10mM) was added, and the volume was brought up to 40μL with DEPC water. The reaction was then carried out at 37℃ for 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h. Figure 8 As shown, the fluorescence signal gradually increases with the extension of reaction time. When the reaction time is 4 hours, the fluorescence signal reaches stability. Therefore, in this embodiment, a reaction time of 4 hours is selected for subsequent research.

[0062] (4) Optimization of reaction temperature To ensure rapid and efficient telomerase extension and DNAzyme digestion, exploring the effect of reaction temperature is crucial. Similarly, the reaction system was first denatured at 95°C for 6 min by a mixture of TP (0.1 μM), L1 (0.1 μM), L2 (0.05 μM), L3 (0.05 μM), S6 (0.1 μM), Tris-HCl (50 mM, pH=7.5), and NaCl (50 mM), followed by natural cooling to room temperature, thus allowing self-assembly to form TSDSD. Subsequently, telomerase extract (TE, 10...) was added to the reaction solution. 4 cells), Sub-FQ (0.1 μM) and Mg 2+ (10mM) was added, and the volume was brought up to 40μL with DEPC water. The reaction was carried out for 4 hours at temperatures of 4℃, 10℃, 25℃, 37℃, 45℃, and 55℃. Figure 9 As shown, the fluorescence value of the system does not change much in the range of 4℃ to 55℃, proving that the strategy can detect telomerase in a wide temperature range. Based on the telomerase detection strategy of the present invention, it is expected to be applied to the detection and analysis of telomerase in cells. Therefore, 37℃ is selected as the optimal temperature for subsequent research in this embodiment.

[0063] (5) Optimization of pH value of the system To ensure the rapid and efficient progress of the reaction under suitable acid-base conditions, exploring the effect of pH on the reaction system is crucial. Similarly, the above reaction system was placed in Tris-HCl buffer solutions with pH values ​​of 5.5, 6.5, 7.5, 8.5, and 9.5, respectively, and reacted at 37°C for 4 hours. The results are as follows... Figure 10 As shown, the fluorescence value of the system does not change much at various pH values, proving that this strategy can detect telomerase over a wide pH range. Based on the telomerase detection strategy of this invention, it is expected to be applied to the detection and analysis of telomerase in cells. Therefore, pH 7.5 was selected as the optimal pH value for subsequent studies in this embodiment.

[0064] (6) Primer concentration optimization To ensure sufficient binding of telomerase to the primers and efficient extension reaction, generating enough extension product to specifically hybridize with the S6 strand, thereby releasing the S6 strand through strand displacement and allowing the substrate strand to bind to the splitting DNA enzyme and activate its cleavage activity, exploring the effect of primer concentration on this cascade reaction is crucial. The above reaction system was placed in reaction solutions with TP primer concentrations of 0, 50, 100, 200, and 500 nM, and reacted at 37°C for 4 h. The results are as follows: Figure 11 As shown, the fluorescence values ​​of the system are the highest and similar at 100 nM and 200 nM, and then decrease. Therefore, 100 nM was selected as the optimal primer concentration for subsequent studies.

[0065] Based on the optimization results of (1) to (6) above, the optimal reaction conditions for this method are determined to be: using Mg 2+ The catalytic metal ion concentration was 10 mM; the reaction time was 4 h; the reaction temperature was 37 °C; the system pH was 7.5; and the primer concentration was 100 nM. Under these conditions, the reaction system composition was: TP (0.1 μM), L1 (0.1 μM), L2 (0.05 μM), L3 (0.05 μM), S6 (0.1 μM), Sub-FQ (0.1 μM), Mg 2+ The solution was prepared with 10 mM Tris-HCl (50 mM, pH 7.5) and 50 mM NaCl, and then brought to a final volume of 40 μL with DEPC water. Subsequent specificity studies, standard curve establishment, interference resistance studies, spiked recovery, and actual sample analysis were all conducted under these optimal conditions.

[0066] The general detection steps based on the above reaction system are as follows: Add the sample to be tested (such as telomerase extract, TE, 10) to the above reaction system. 4 The cells were reacted at 37°C for 4 hours, and the fluorescence intensity of the system at 520 nm was detected.

[0067] III. Specificity of Telomerase Detection To verify whether this method specifically detects telomerase without interference from other endogenous enzymes, it is crucial to explore the effects of different endogenous proteases on the signal amplification reaction of the system. The above reaction system was then subjected to reactions in mixtures of different endogenous proteases (APE1, Trypsin, DNMT1, EXO I, and TE) at the same concentration. Figure 12 Only telomerase can trigger the elongation and cleavage reactions, producing a significant fluorescence signal intensity. Other endogenous proteases cannot trigger this reaction in the system, so the fluorescence signal value is close to that of the blank group. In summary, this study's strategy can efficiently and specifically detect telomerase.

[0068] IV. Establishment of the Standard Curve Under optimal conditions, the reaction system was placed in telomerase extract solutions containing a series of different numbers of HeLa cells, with the reaction conditions as before. The fluorescence intensity at 520 nm of the reaction system after adding telomerase extract was defined as F, and the fluorescence intensity of the blank group (without telomerase extract or with heat-inactivated telomerase extract) under the same conditions was defined as F0. The number of HeLa cells was defined as N, and (F-F0) / F0 was plotted against N. Figure 13 As shown, the fluorescence value increases with the increase of cell number, and tends to stabilize when the cell number reaches 2000 cells. Therefore, the detection strategy has a good linear relationship with the fluorescence value in HeLa cells between 20-1800 cells, and the linear regression equation is (F-F0) / F0=0.00339N+0.214, R. 2 =0.99, LOD=12 cells / 40uL (<1 cell / uL), indicating that the strategy has high telomerase sensitivity.

[0069] V. Anti-interference capability assessment To evaluate the selectivity of this detection method in real-world complex biological samples, it is crucial to examine the reaction system's resistance to potential interfering substances. Several experimental groups were set up, one with interfering substances (GSH, ATP, BSA, FBS) and the other without. The differences in fluorescence values ​​between the groups were compared, while the proportions of other raw materials in the system remained the same as before. Figure 14As shown, in the presence of telomerase, the fluorescence values ​​of the groups with and without the interfering agent were relatively stable and significantly higher than those of the blank group, indicating that the system has good anti-interference ability in relevant biological matrices.

[0070] VI. Inspection of Spiked Recycling To verify the accuracy and reliability of this detection method in the analysis of actual biological samples, it is crucial to examine its spiked recovery capability for telomerase. Telomerase extracts with three different cell numbers were prepared and added to reaction systems with and without serum, respectively. All ingredient ratios and reaction conditions remained the same as before. Fluorescence intensity values ​​at 520 nm were recorded and substituted into the standard curve to calculate the recovery rate. The results are shown in Tables 2 and 3. The spiked recoveries of each group were within a reasonable range (96%–101%), and the relative standard deviations were small (1.113%–7.664%), indicating that the detection method has good accuracy and precision and can meet the needs of telomerase activity detection in actual biological samples.

[0071] Table 2. Spiked Recovery (Serious-Free)

[0072] Table 3. Spiked Recovery (including serum)

[0073] VII. Detection of actual target samples To verify the applicability and reliability of this detection method in actual biological samples, telomerase activity was detected in cell lysate samples and clinical tissue samples, respectively.

[0074] (1) Telomerase detection in cell lysate samples This system was used to detect telomerase in cell lysates obtained from cervical cancer cells (HeLa), human lung adenocarcinoma cells (A549), human breast cancer cells (MCF-7), human chronic myeloid leukemia cells (K562), human liver cancer cells (SMMC-7721), and human embryonic kidney cells (HEK293T), as well as in HeLa lysates (EGCG) treated with telomerase inhibitors. The reaction system was the same as before. A mixture of TP (0.1 μM), L1 (0.1 μM), L2 (0.05 μM), L3 (0.05 μM), S6 (0.1 μM), Tris-HCl (50 mM, pH=7.5), and NaCl (50 mM) was denatured at 95 °C for 6 min, then naturally cooled to room temperature to self-assemble into TSDSD. Telomerase extract (TE, 10...) was then added to the reaction solution. 4 Actual sample (cells), Sub-FQ (0.1 μM) and Mg 2+Add 10mM DEPC water to a final volume of 40μL, and perform extension and digestion reactions at 37℃ for 4 hours. Figure 15 The results showed that telomerase was highly expressed in HEK293T cells and various cancer cells, consistent with relevant literature reports. The fluorescence intensity of the EGCG group containing a telomerase inhibitor was significantly reduced, indicating that this method can effectively distinguish different telomerase expression levels. Parallel control experiments using a traditional ELISA kit showed that the relative expression levels in each group were almost identical, demonstrating the accuracy and reliability of this method in detecting telomerase activity in actual cell lysate samples.

[0075] (2) Telomerase detection in clinical tissue samples To further evaluate the diagnostic applicability and reliability of this detection method in actual clinical samples, this method was used to detect telomerase activity in clinical tissue samples. The system was used to detect telomerase activity in normal tissue samples from a healthy control group (7 cases) and cancerous tissue samples from cervical cancer patients (8 cases), with the reaction system and conditions as before, and fluorescence signal detection was performed. Parallel control experiments were also conducted using the traditional ELISA method. Compared with the results from the ELISA kit, such as... Figure 16 B and Figure 16 As shown in F, box plot analysis revealed that the median telomerase activity (characterized by (F-F0) / F0) in the Cancer group was significantly higher than that in the Healthy group (p<0.01), and the data distributions of the two groups did not significantly overlap; heatmap ( Figure 16 Further analysis (C and 16G) showed that all healthy tissue samples exhibited low signal responses (blue areas), while cancer tissue samples generally showed high signal responses (red areas). The intra-group consistency was good, and the inter-group differentiation was significant, indicating that this method has good discriminative ability for telomerase activity in clinical tissue samples. In addition, the receiver operating characteristic (ROC) curve analysis results ( Figure 16 D) The ability of this method to distinguish between cervical cancer tissue and healthy tissue was further evaluated. Figure 16 As shown in Figure D, the area under the ROC curve (AUC) of the method in this study is 0.96, which is significantly better than the AUC (0.80) of the traditional ELISA method. It has higher sensitivity and specificity, and the overall diagnostic efficacy reaches an excellent level, which can provide a more reliable detection basis for the clinical auxiliary diagnosis of cervical cancer.

[0076] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0077] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for detecting telomerase activity, characterized in that, Includes the following steps: A functional DNA module is provided, the functional DNA module comprising a telomerase primer and a blocking splitting DNAzyme, the telomerase primer being linked to the blocking splitting DNAzyme, the blocking splitting DNAzyme comprising a DNAzyme nucleic acid strand and at least two blocking strands, the DNAzyme nucleic acid strands assembling to form a splitting DNAzyme catalytic core, the at least two blocking strands hybridizing with the DNAzyme nucleic acid strand respectively to inhibit the catalytic activity of the DNAzyme nucleic acid strand; The sample to be tested is mixed with the functional DNA module, so that the telomerase in the sample to be tested recognizes and extends the telomerase primer, generating an extension product containing telomere repeat sequences. The extended product replaces the closed strand from the closed split DNAzyme through a chain substitution reaction, thereby restoring the catalytic activity of the DNAzyme nucleic acid chain; The DNAzyme nucleic acid strand cleaves the substrate strand and generates a detectable signal; The detectable signal is detected, and the activity of telomerase in the sample to be tested is determined based on the intensity of the detectable signal.

2. The detection method according to claim 1, characterized in that, The DNAzyme nucleic acid chain is a splitting DNAzyme nucleic acid chain based on an 8-17 DNAzyme catalytic core. The DNAzyme nucleic acid chain recognizes the rAG site in the substrate chain and cleaves the substrate chain at that site.

3. The detection method according to claim 1, characterized in that, The reaction in which the DNAzyme nucleic acid chain cleaves the substrate chain is carried out in the presence of a divalent metal ion, namely a magnesium ion.

4. The detection method according to claim 1, characterized in that, The substrate chain is modified with a fluorescent group and a fluorescence quenching group at both ends, and the substrate chain generates a fluorescence signal after being cleaved; the fluorescent group is FAM and the fluorescence quenching group is BHQ1.

5. The detection method according to claim 1, characterized in that, The blocked split DNAzyme comprises a DNAzyme nucleic acid chain and two blocking chains. The DNAzyme nucleic acid chain includes three nucleic acid chains. The two blocking chains hybridize and assemble with the three nucleic acid chains to achieve double blocking of the blocked split DNAzyme.

6. The detection method according to claim 1, characterized in that, The sample to be tested is a cell lysate, tissue sample, blood sample, or clinical biopsy sample.

7. The detection method according to claim 1, characterized in that, The step of determining the telomerase activity in the sample to be tested based on the intensity of the detectable signal includes: comparing the intensity of the detectable signal with a pre-established standard curve to calculate the number of telomerase-positive cells in the sample to be tested; the standard curve has the number of telomerase-positive cells as the abscissa and the intensity of the detectable signal as the ordinate.

8. A telomerase activity detection kit, characterized in that, Include: A functional DNA module comprising a telomerase primer and a blocking splitting DNAzyme, wherein the telomerase primer is attached to the blocking splitting DNAzyme, the blocking splitting DNAzyme comprises a DNAzyme nucleic acid strand and at least two blocking strands, the DNAzyme nucleic acid strands assembling to form a splitting DNAzyme catalytic core, and the at least two blocking strands hybridizing with the DNAzyme nucleic acid strands respectively to inhibit the catalytic activity of the DNAzyme nucleic acid strands; A substrate chain that can be cleaved by a catalytically active DNAzyme nucleic acid chain; Reaction buffer.

9. The detection kit according to claim 8, characterized in that, The detection kit is used for in vitro telomerase activity detection or for in situ imaging analysis of intracellular telomerase activity.

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