Novel method for thrombin detection through enhanced entropy-driven DNA non-covalent catalytic reaction based on paired joint structure design

Abstract

The invention relates to a thrombin detection technology, in particular to an enhanced entropy-driven DNA (deoxyribonucleic acid) non-covalent catalytic reaction based on paired joint structural design, which is used for sensitive and specific detection of thrombin. According to the method, a target-specific aptamer is designed, a thrombin target is recognized through a proximity ligation strategy, and finally the enhanced entropy-driven DNA non-covalent catalytic reaction is triggered.

Description

Technical Field

[0001] The present invention relates to a thrombin detection technology, and in particular to an enhanced entropy-driven DNA non-covalent catalytic reaction based on paired linker structure design, which is used for sensitive and specific detection of thrombin. Background Art

[0002] DNA has become a core material in multiple fields, including diagnostics, therapeutics, molecular computing, and nanodevices. The application of DNA in nanotechnology is largely due to its predictable Watson-Crick base pairing and our comprehensive understanding of its hybridization thermodynamics and kinetics. Toehold-mediated strand displacement (TMSD) and toehold exchange, processes in which the invading strand competitively displaces one strand of the DNA duplex by forming base pairs, have facilitated the development of dynamic DNA systems. Although continuously operating dynamic systems, including reaction network structures and synthetic molecular machines, have been developed, the state-of-the-art technology has not yet reached a level of functionality and flexibility that matches their natural counterparts.

[0003] Catalytic reactions play an important role in chemical and biological systems and are the basis for a wide range of RNA and protein enzyme activities. In engineered molecular systems, DNA has also been shown to perform non-covalent catalytic reactions, which provides the potential for embedding more complex control in chemistry, materials, and medicine. For example, DNA catalysts can be used for signal amplification in molecular diagnostics and can be combined with other structures to perform various functions, including signal recovery and weighted multiplication. Turberfield et al. first constructed a non-enzymatic catalytic system that opens the ring through toehold-initiated strand displacement, thereby increasing the reaction rate. Seelig et al. improved this system to make it more stable and efficient. The catalysts in these systems can be regarded as prototype molecular motors because they undergo conformational changes from single-stranded DNA to double-stranded DNA, and in theory this movement can drive nanomachines.

[0004] To meet this challenge, the present invention utilizes the high specificity of DNA strand displacement and develops a paired joint structure (PJS) strategy to enhance the thermodynamic driving force of DNA strand displacement reactions. Two complementary single-stranded arms are inserted into the substrate of the non-covalent DNA catalytic reaction to increase the system entropy while minimizing the effect on the reaction rate and maintaining low leakage. The introduction of PJS increases the catalytic reaction yield of the reaction substrate because the reaction is transformed into a process of increasing entropy, which has a higher thermodynamic driving force and is easier to occur and proceed comprehensively. More importantly, the system has also been used to construct a biosensing platform to directly detect thrombin in a complex biological environment, demonstrating its robustness and sensitivity. Summary of the invention

[0005] The purpose of the present invention is to develop a simple, sensitive and specific thrombin detection strategy.

[0006] The specific technical solutions are as follows:

[0007] A novel method for detecting thrombin based on an enhanced entropy-driven DNA non-covalent catalytic reaction designed based on a paired linker structure, wherein the detection method comprises the following steps:

[0008] (1) Annealing of reaction substrate: The reaction substrate is heated at 95°C for 5 min, then decreased to 25°C at a rate of 0.1°C per second and maintained at that temperature for 5 min. It is then placed at 4°C for later use.

[0009] (2) Fluorescence detection process In order to detect different concentrations of thrombin, the solution in part A consists of substrate 1 and substrate 2. The solution in part B consists of thrombin aptamer 1, thrombin aptamer 2, and TE Mg 2+ First, the B part solution was incubated at room temperature for 5 minutes. Subsequently, the A part and the B part were mixed at 37°C, and the fluorescence intensity was monitored by a Rotor-Gene Q real-time fluorescence quantitative PCR analyzer (QIAGEN, Germany).

[0010] The concentration of substrate 1 in step (2) is preferably 250 nM.

[0011] The concentration of substrate 2 in step (2) is preferably 250 nM.

[0012] The concentration of aptamer 1 in step (2) is preferably 200 nM.

[0013] The concentration of aptamer 2 in step (2) is preferably 200 nM.

[0014] In the step (2), TE Mg 2+ The best concentration is 12.5mM / L.

[0015] The present invention uses a paired joint structure (PJS) strategy to establish a specific and sensitive thrombin detection method. Since thrombin has two specific aptamers that bind to different epitopes, and each aptamer is extended by a split catalyst C containing a short complementary sequence in the middle, they can bind to each other. The detection principle is that when the target thrombin coexists with the two aptamers, the aptamer will bind to the thrombin and cause a proximity effect, thereby forming an active reconstructed catalyst C, which will eventually trigger the constructed enhanced entropy-driven DNA non-covalent catalytic reaction (PJS-EDCR). For PJS-EDCR, the specific principle is as follows (Appendix Figure 1): Under the action of catalyst C, C first replaces the three-chain complex (PS1_2). Subsequently, F1_2 replaces C again through reverse toehold, and then undergoes four-way branch migration, eventually producing two types of products: S1F1 and S2F2 with significant fluorescence changes.

[0016] PAGE analysis was first used to verify the PJS-EDCR reaction process (Appendix Figure 2 ). First, in lane 6, there is a mixture of two reaction substrates, but no new bands are observed, indicating that the two reaction substrates of PJS-EDCR remain stable and do not react with each other in the absence of catalyst C. When the C chain is added to the pure S1_2 substrate, no new bands appear because no fuel chain is added to the reaction and the cyclic reaction does not occur (lane 5). When the C chain is added to the pure S1_2 substrate, no new bands appear because no fuel chain is added to the reaction and the cyclic reaction does not occur. The above results strongly confirm the successful design and feasibility of PJS-EDCR.

[0017] In order to further analyze the PJS-EDCR reaction process, we conducted fluorescence kinetics experiments (see Appendix Figure 3 ). In the absence of catalyst C, the fluorescence intensity of the blank control system barely increased, confirming the ability of PJS-EDCR to maintain low leakage. However, in the presence of catalyst C, the fluorescence intensity increased rapidly, indicating the occurrence of PJS-EDCR. The above results indicate that we have successfully constructed a novel enzyme-free DNA circuit signal amplification system that can be applied to the high-sensitivity and high-specificity detection of thrombin.

[0018] After establishing the thrombin-responsive PJS-EDCR (Appendix Figure 4 ), we further evaluated the sensitivity of the sensor to thrombin. Thrombin at different concentrations ranging from 1 nM to 250 nM was added to the reaction system at 37 °C for 1 hour (see Figure 5 By real-time monitoring of the thrombin-responsive PJS-EDCR, we observed that the fluorescence signal showed a good linear relationship with the thrombin concentration in the range of 1 nM to 50 nM ((Appendix Figure 6 ). The calibration equation is Y = 0.19958 + 0.00946X, and the correlation coefficient R2 is 0.9914. The detection limit (LOD) is calculated to be 0.2nM. Compared with other thrombin-responsive enzyme-free DNA circuit signal amplification systems, HEAR can significantly improve sensitivity and convenience.

[0019] In order to evaluate the specificity of the constructed thrombin-responsive PJS-EDCR assay, we introduced some related proteins (LgG, fibrinogen, bovine serum albumin (BSA), recombinant albumin (ALB), and myoglobin (MYO)). At the same concentration, the signal induced by thrombin molecules was significantly higher than that induced by other interfering substances or similar compounds (see Appendix). Figure 7 ). This confirms that this detection method has satisfactory selectivity. The above data prove that this design has excellent protein sensing performance.

[0020] This patent proposes a new strong entropy-driven DNA non-covalent catalytic reaction strategy for high-sensitivity and high-specificity detection of thrombin. The thrombin-responsive PJS-EDCR only requires two simply designed reaction substrates and two aptamers to quickly and easily detect thrombin in one pot. The method has high sensitivity and high specificity, with a detection limit of 0.2nM. The results show that the thrombin-responsive PJS-EDCR method has great potential in disease diagnosis and biomedical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of PJS-EDCR

[0022] Figure 2 PAGE characterization of PJS-EDCR

[0023] Figure 3 Fluorescence kinetics characterization of PJS-EDCR

[0024] Figure 4 Schematic diagram of thrombin-responsive PJS-EDCR

[0025] Figure 5 Real-time fluorescence kinetics of thrombin at different concentrations

[0026] Figure 6 Linear fit between fluorescence response and thrombin concentration.

[0027] Figure 7 Analysis of fluorescence changes of thrombin, IgG, fibrinogen, bovine serum albumin (BSA), recombinant albumin (ALB) and myoglobin (MYO) with thrombin-responsive PJS-EDCR. DETAILED DESCRIPTION

[0028] A simple, sensitive and specific strategy for detecting trace amounts of thrombin is as follows:

[0029] (1) Annealing of reaction substrate: The reaction substrate is heated at 95°C for 5 min, then decreased to 25°C at a rate of 0.1°C per second and maintained at that temperature for 5 min. It is then placed at 4°C for later use.

[0030] (2) Fluorescence detection process In order to detect different concentrations of thrombin, the solution in part A consists of substrate 1 and substrate 2. The solution in part B consists of thrombin aptamer 1, thrombin aptamer 2, and TE Mg 2+ First, the B part solution was incubated at room temperature for 5 minutes. Subsequently, the A part and the B part were mixed at 37°C, and the fluorescence intensity was monitored by a Rotor-Gene Q real-time fluorescence quantitative PCR analyzer (QIAGEN, Germany).

Claims

1. A method for sensitive and specific detection of trace nucleic acids based on a hairpin probe-mediated exponential amplification reaction, characterized in that The steps include: (1) Annealing of reaction substrate: The reaction substrate is heated at 95°C for 5 min, then decreased to 25°C at a rate of 0.1°C per second and maintained at 25°C for 5 min, and then placed at 4°C for later use; (2) Fluorescence detection process To detect different concentrations of thrombin, the solution in part A consists of substrate 1 and substrate 2; the solution in part B consists of thrombin aptamer 1, thrombin aptamer 2, and TE Mg 2+ Composition: First, incubate the solution of Part B at room temperature for 5 minutes; Subsequently, part A and part B were mixed at 37°C, and the fluorescence intensity was monitored by Rotor-Gene Q real-time fluorescence quantitative PCR analyzer (QIAGEN, Germany).

2. The circulation method using two reaction substrates and two aptamers as claimed in claim 1.

3. A novel method for detecting thrombin based on an enhanced entropy-driven DNA non-covalent catalytic reaction designed based on a paired linker structure as described in claim 1, wherein the concentration of substrate 1 in step (2) of the preparation method is 1 μM-100 nM.

4. A novel method for detecting thrombin based on an enhanced entropy-driven DNA non-covalent catalytic reaction designed based on a paired linker structure as claimed in claim 1, wherein the concentration of substrate 2 in step (2) of the preparation method is 1 μM-100 nM.

5. A novel method for detecting thrombin based on an enhanced entropy-driven DNA non-covalent catalytic reaction designed based on a paired linker structure as claimed in claim 1, wherein the concentration of aptamer 1 in step (2) of the preparation method is 1 μM-100 nM.

6. A novel method for detecting thrombin based on an enhanced entropy-driven DNA non-covalent catalytic reaction designed based on a paired linker structure as claimed in claim 1, wherein the concentration of aptamer 2 in step (2) of the preparation method is 1 μM-100 nM.

7. A novel method for detecting thrombin based on an enhanced entropy-driven DNA non-covalent catalytic reaction designed based on a paired linker structure as claimed in claim 1, wherein in step (2) of the preparation method, TE Mg 2+ The concentration is 50mM / L-1mM / L.

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