Method for detecting miR-21 based on DNA tetrahedrons with different functions and spatial local cascade cycle catalytic amplification strategy

Abstract

The invention relates to a method for detecting microRNA-21 (miR-21) based on DNA tetrahedrons with different functions and a spatial local cascade cycle catalytic amplification strategy. According to the method, two kinds of DNA tetrahedrons (a catalytic DNA tetrahedron Cat-DT and a signal DNA tetrahedron Sig-DT) with different functions are designed, and a cascade cycle amplification circuit is constructed. The miR-21 can trigger the release of an enzyme chain (E-DNA), the Cat-DT is converted into a three-branch DNA nanostructure (TB-DNA), each TB-DNA carries three E-DNA units, the conversion of more Cat-DT is further catalyzed, and the exponential amplification is realized. Meanwhile, TB-DNA and Sig-DT interact to generate a fluorescence signal, and a miR-21 simulation chain is regenerated to form a circulation mechanism, so that the reaction efficiency is remarkably improved. According to the method, the detection linear range is 600 fM to 100 nM, the detection limit is as low as 400 fM, and the total reaction time is shortened to 80 minutes. In addition, the method has good practical application capability and wide application prospect in the aspect of miR-21 detection.

Description

Technical Field

[0001] This invention relates to biological detection technology, and in particular to a highly sensitive method for detecting miRNA based on DNA tetrahedral nanostructures with different functions and a cascade catalytic amplification strategy, for achieving specific and highly sensitive detection of miR-21. Background Technology

[0002] MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs, approximately 19-23 nucleotides in length, that regulate gene expression by binding to target mRNAs and play a crucial role in tumorigenesis and development. miR-21, an oncogene miRNA, is significantly highly expressed in prostate cancer tissues, serum, and urine, making it an important biomarker. Currently, miRNA detection methods such as RT-PCR, FISH, and ddPCR suffer from drawbacks such as high sample consumption, poor specificity, and complex operation. To improve sensitivity, nucleic acid isothermal amplification techniques such as hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) are widely used; however, traditional methods rely on linear amplification, require constant catalyst concentration, have limited amplification efficiency, and have long reaction times. Summary of the Invention

[0003] The present invention aims to provide a highly sensitive and rapid method for detecting microRNA-21 (miR-21) to solve the technical problems of slow reaction kinetics and limited detection sensitivity that are common in existing detection technologies.

[0004] To achieve the above objectives, the present invention employs a miR-21 detection method based on a spatially localized cascaded cyclic catalytic amplification strategy. The core of this method lies in constructing a cascaded cyclic amplification circuit using DNA tetrahedra with different functions—catalytic DNA tetrahedra (Cat-DT) and signal DNA tetrahedra (Sig-DT). miR-21 triggers locked-E-DNA to release E-DNA, which converts Cat-DT into TB-DNA. Each TB-DNA carries three E-DNA units, exponentially catalyzing the conversion of more Cat-DT. Simultaneously, TB-DNA interacts with Sig-DT to generate a fluorescent signal and regenerates the miR-21 mimic strand, thus forming a second-stage cyclic amplification, achieving continuous and efficient signal enhancement.

[0005] Compared with the prior art, the present invention has the following beneficial effects: the method significantly improves reaction kinetics and detection sensitivity through the synergistic effect of spatial localization and cyclic amplification, shortens the total reaction time to 80 minutes, and achieves a detection limit of 400 fM; it exhibits a good linear relationship in the concentration range of 600 fM to 100 nM and shows excellent specificity, demonstrating good potential for practical application.

[0006] Specifically, this invention provides a method for detecting miR-21 based on a target-induced cascaded catalytic amplification (TICA) strategy using different functional DNA tetrahedra. This method constructs a cascaded cyclic amplification circuit by designing catalytic DNA tetrahedra (Cat-DT) and signal DNA tetrahedra (Sig-DT) to achieve high-sensitivity detection. The method includes the following steps: 1. DNA tetrahedron preparation steps: Preparation of catalytic DNA tetrahedrons (Cat-DT): Dissolve sequences Cat-1, Cat-2, Cat-3 and S-DNA in equimolar ratio in 10 mM PBS buffer (pH 7.5, containing 50 mM NaCl), heat to 90°C and hold for 5 minutes, then slowly cool to room temperature. Subsequently, add S-DNA at a molar concentration three times that of Cat-1 and incubate at 35°C for 15 minutes to form Cat-DT.

[0007] Preparation of signal DNA tetrahedra (Sig-DT): Sequences Sig-1, Sig-2, Sig-3, and T were treated in equal molar ratios, heated and cooled, and then T was added at a molar concentration three times that of Sig-1. The mixture was incubated at 35°C for 15 minutes to form Sig-DT. The ends of Sig-1, Sig-2, and Sig-3 were modified with the fluorescent group FAM and the quencher group BHQ1, forming a fluorescence quenching system.

[0008] Key sequences: miR-21: 5'-UAGCUUAUCAGACUGAUGUUGA-3' Cat-1: 5'-ATCAGAAGCGATTAACCACACTTCAAACTTCAAAGTTTTATGGAAGGGTTACACCCATGTTAGCTT-3' Cat-2: 5'-ATCAGAAGCGATTAACCACTTCCATAAAACAAAAGTGCCGCGTTTGGTTACACCCATGTTAGCTT-3' Cat-3: 5'-ATCAGAAGCGATTAACCAAAACGCGGCACTAAAGAAGTTGAAGTGGGTTACACCCATGTTAGCTT-3' S-DNA: 5'-CTTCTGATAAAAGCTArATCTGATTTAAGCTAAC-3' (rA represents ribonucleotide) Sig-1: 5'-FAM-AAGCTAACATCACTTCAAACTTCAAAGTTTTATGGAAGCGCTTCTGAT-BHQ1-3' Sig-2: 5'-FAM-AAGCTAACATCTTCCATAAAACAAAAGTGCCGCGTTTCGCTTCTGAT-BHQ1-3' Sig-3: 5'-FAM-AAGCTAACATAAACGCGGCACTAAAGAAGTTGAAGTGCGCTTCTGAT-BHQ1-3' T: 5'-TAGCTTATCAGACTGATGTTGA-3' Locked-E-DNA: 5'-ATCAGAAGCGATTAACCAGGTTACACCCATGTTAGCTT-3' (used to trigger E-DNA release) 2. Detection reaction steps: The miR-21 sample to be tested was mixed with Locked-E-DNA and added to a reaction solution containing 150 nM Cat-DT and 150 nM Sig-DT. The reaction was carried out in 10 mM PBS buffer (pH 7.5, containing 100 mM NaCl and 20 mM MgCl2) and incubated at 37°C for 80 minutes, triggering a cascaded catalytic amplification reaction. miR-21 competitively binds to and releases E-DNA, which catalyzes the conversion of Cat-DT to three-branched TB-DNA. TB-DNA interacts with Sig-DT to generate a fluorescent signal and regenerates the miR-21 mimic strand, forming a cyclic amplification process.

[0009] 3. Fluorescence detection steps: The fluorescence spectrum of the reaction solution was measured with an excitation wavelength of 486 nm and an emission spectrum scanned in the range of 505-600 nm, with a focus on measuring the fluorescence intensity at 520 nm. The concentration of miR-21 in the sample was calculated using the standard curve method (calibration equation: F = 166.2 lg CmiR-21 + 707.0, linear range 600 fM-100 nM).

[0010] Preferred implementation scheme: In step 1), the annealing process for Cat-DT and Sig-DT needs to be strictly controlled to ensure the stability of the tetrahedral structure; the final concentration of Cat-DT and Sig-DT is optimized to 150 nM to balance the signal intensity and background noise.

[0011] In step 2), the reaction buffer should contain 20 mM MgCl2 to activate DNAzyme activity, and an incubation time of 80 minutes can ensure complete reaction and avoid nonspecific amplification.

[0012] In step 3), fluorescence detection should be performed immediately after the reaction to reduce signal attenuation; the standard curve should be plotted using freshly prepared miR-21 standards to ensure accuracy.

[0013] This method achieves highly efficient concentration of catalysts and reactants through spatial localization design, with a detection limit of 400 fM and a reaction time shortened to 80 minutes, demonstrating high practicality and specificity in real-world applications. Attached Figure Description

[0014] Figure 1 The diagram shows the detection principle of the present invention: (A) the logical flow of the cascaded cyclic amplification strategy; (B) the detailed reaction steps of the DNA tetrahedron based on the TICA strategy, including miR-21 triggering E-DNA release, Cat-DT conversion to TB-DNA, and the interaction between TB-DNA and Sig-DT.

[0015] Figure 2 Fluorescence spectra of samples under different conditions: (1) Blank sample, (2) Locked-EDNA, (3) Locked-EDNA+Cat-DT, (4) Locked-EDNA+Cat-DT+Sig-DT, (5) Locked-EDNA+Cat-DT+Sig-DT+miR-21.

[0016] Figure 3 The results of the selective study of miR-21 and other similar sequences using the method of this invention are shown in the figure. The composition of the six samples is as follows: blank sample, miR-16, miR-92a, miR-146a, miR-21 and mixed sample (the mixed sample contains miR-21 and other miRNA sequences). Detailed Implementation

[0017] The present invention will be further described in detail below with reference to embodiments. The preferred embodiment of the method of the present invention is as follows: 1. DNA tetrahedron preparation steps: Cat-DT preparation: Dissolve sequences Cat-1, Cat-2, and Cat-3 in 1:1:1 molar ratio in 10 mM PBS buffer (pH 7.5, containing 50 mM NaCl), heat to 90°C and hold for 5 minutes, then slowly cool to room temperature. Add S-DNA (molar concentration three times that of Cat-1) and incubate at 35°C for 15 minutes to form Cat-DT.

[0018] Preparation of Sig-DT: Sequences Sig-1, Sig-2, and Sig-3 were treated in the same molar ratio of 1:1:1, heated and cooled, and then T (molar concentration three times that of Sig-1) was added. The mixture was incubated at 35°C for 15 minutes to form Sig-DT. In Sig-DT, the fluorescent group FAM is close to the quenching group BHQ1, and the fluorescence signal is quenched.

[0019] 2. Detect miR-21: The miR-21 sample to be tested was mixed with Locked-E-DNA, and a reaction solution containing 150 nM Cat-DT and 150 nM Sig-DT (10 mM PBS buffer, pH 7.5, containing 100 mM NaCl and 20 mM MgCl2) was added. The mixture was incubated at 37°C for 80 minutes. miR-21 competitively binds to trigger the release of E-DNA. E-DNA catalyzes the conversion of Cat-DT to TB-DNA. TB-DNA reacts with Sig-DT to generate a fluorescent signal and regenerates the miR-21 mimic strand, achieving cyclic amplification.

[0020] 3. Fluorescence detection: A fluorescence spectrophotometer was used, with excitation wavelength of 486 nm and emission wavelength scanning from 505 to 600 nm. The fluorescence intensity at 520 nm was measured. The result was expressed as the logarithm of miR-21 concentration (lg...). CmiR-21 Plot a standard curve with fluorescence intensity as the ordinate and fluorescence denoted by π / 2. For example, the equation is F = 166.2 lg C. miR-21 +707.0 (linear range 600 fM-100 nM), calculate sample concentration.

[0021] The above method is the preferred embodiment of the present invention.

[0022] miR-21 detection principle of the present invention like Figure 1As shown in Figure B, miR-21 competitively binds to trigger the release of the enzyme chain (E-DNA) from locked-E-DNA, activating a Mg²⁺-specific DNAzyme. The released E-DNA interacts with the catalytic DNA tetrahedron (Cat-DT) to form a functional DNAzyme, which cleaves the S-DNA on the Cat-DT, converting the Cat-DT into a three-branched DNA nanostructure (TB-DNA). Each TB-DNA carries three E-DNA units, further catalyzing the conversion of more Cat-DT, achieving exponential amplification. Subsequently, the TB-DNA reacts with the signal DNA tetrahedron (Sig-DT), triggering the generation of a fluorescence signal. Crucially, this process simultaneously regenerates the miR-21 mimic strand, forming a cyclic amplification mechanism. Through this spatially localized design, the effective concentrations of the catalyst and reactants are significantly increased, resulting in a substantial improvement in reaction efficiency.

[0023] Feasibility characterization of the method To verify the feasibility of the strategy, a control group experiment was designed. For example... Figure 2 As shown, the blank control (sample 1) was used to subtract background fluorescence and establish a signal baseline. The basal treatment group (sample 2, Locked-E-DNA) contained only the locked enzyme-DNA complex, used to confirm that the system itself did not produce a significant fluorescence signal in the absence of a trigger signal. The Cat-DT treatment group (sample 3, Locked-E-DNA + Cat-DT) added catalytic DNA to the basal group to verify that Cat-DT itself would not nonspecifically activate the fluorescence signal under nonspecific triggering conditions. The complete system without miR-21 (sample 4, Locked-E-DNA + Cat-DT + Sig-DT) constructed a complete reaction system excluding the target miRNA (miR-21). As shown in the figure, this sample only produced a weak fluorescence signal, clearly indicating that the cascade reaction could not be effectively initiated in the absence of miR-21, thus conversely proving that miR-21 is an essential factor in triggering the entire reaction. The complete system containing miR-21 (Sample 5, Locked-E-DNA + Cat-DT + Sig-DT + miR-21): This system added the target miR-21 to Sample 4. Results showed a significant enhancement in fluorescence signal compared to Sample 4. Quantitative comparison revealed that the cyclic amplification system composed of Cat-DT and Sig-DT exhibited 1.6 times the fluorescence enhancement effect without this cyclic system, directly demonstrating the effectiveness of the cyclic amplification mechanism.

[0024] The selectivity of miR-21 in this invention The selectivity of this method was evaluated by comparing the responses of miR-21 with heterologous miRNAs (such as miR-16, miR-92a, and miR-146a). Figure 3 As shown, the system produces a significant fluorescent signal only in the presence of miR-21, while the signals of other miRNAs are similar to those of the blank control. Mixed samples (containing miR-21 and other miRNAs) also show strong signals, indicating that miR-21 can specifically trigger the cascade reaction and has strong anti-interference capabilities. These results demonstrate that the present invention has high specificity and selectivity for miR-21.

[0025] The practicality of miR-21 detection was assessed by adding different concentrations of miR-21 to 10-fold diluted healthy human serum for recovery experiments. The results showed recoveries ranging from 93.4% to 109.7%, with relative standard deviations (RSDs) of 6.3% to 8.5%. Furthermore, miR-21 levels in patient serum samples were consistent with q-PCR results. These data demonstrate that the present invention exhibits high accuracy and reliability in real serum matrices, meeting practical detection needs.

[0026] In summary, this invention develops a target-induced cascade catalytic amplification (TICA) strategy based on different functional DNA tetrahedra for miR-21 detection. By constructing a cascade catalytic amplification mechanism, the reaction efficiency is significantly improved. This method achieves a detection limit of 400 fM and a wide linear range of 600 fM-100 nM, with the total reaction time reduced to 80 minutes. It exhibits recoveries of 93.4%-109.7% and good specificity in clinical serum samples. Furthermore, this invention provides valuable reference for miRNA detection and disease diagnosis.

Claims

1. A method for detecting miR-21 based on DNA tetrahedrons with different functions and a cascaded cyclic catalytic amplification strategy, comprising the following steps: (1) Preparation of catalytic DNA tetrahedron Cat-DT and signal DNA tetrahedron Sig-DT: DNA sequences Cat-1, Cat-2, Cat-3 and S-DNA were mixed in proportion and Cat-DT solution was formed in 10 mM PBS buffer; DNA sequences Sig-1, Sig-2, Sig-3 and T sequence were mixed in proportion and Sig-DT solution was formed in PBS buffer, wherein Sig-1, Sig-2 and Sig-3 were modified with fluorescent group FAM and quenching group BHQ1 at the end; (2) Mix the miR-21 sample to be tested with Locked-E-DNA, and then mix it with the Cat-DT solution and Sig-DT solution obtained in step 1) to trigger a cascaded catalytic amplification reaction; (3) Detect the fluorescence spectrum of the solution obtained in step 2) and use the standard curve method to determine the concentration of miR-21 in the sample to be tested; in, The miR-21 sequence is 5'-UAGCUUAUCAGACUGAUGUUGA-3', the Locked-E-DNA sequence is 5'-ATCAGAAGCGATTAACCAGGTTACACCCATGTTAGCTT-3', the Cat-1 sequence is 5'-ATCAGAAGCGATTAACCACACTTCAAACTTCAAAGTTTTATGGAAGGGTTACACCCATGTTAGCTT-3', the Cat-2 sequence is 5'-ATCAGAAGCGATTAACCACTTCCATAAAACAAAAGTGCCGCGTTTGGTTACACCCATGTTAGCTT-3', and the Cat-3 sequence is 5'-ATCAGAAGCGATTAACCAAAACGCGGCACTAAAGAAGTTGAAGTGGGTTACAC The S-DNA sequence is 5'-CTTCTGATAAAAGCTArATCTGATTTAAGCTAAC-3' (rA represents ribonucleotide), the Sig-1 sequence is 5'-FAM-AAGCTAACATCACTTCAAACTTCAAAGTTTTATGGAAGCGCTTCTGAT-BHQ1-3', the Sig-2 sequence is 5'-FAM-AAGCTAACATCTTCCATAAAACAAAAGTGCCGCGTTTCGCTTCTGAT-BHQ1-3', the Sig-3 sequence is 5'-FAM-AAGCTAACATAAACGCGGCACTAAAGAAGTTGAAGTGCGCTTCTGAT-BHQ1-3', and the T sequence is 5'-TAGCTTATCAGACTGATGTTGA-3'.

2. The method as described in claim 1, characterized in that... Step 1) Specifically: In the preparation of Cat-DT, Cat-1, Cat-2 and Cat-3 are first mixed in a 1:1:1 molar ratio, heated at 90°C for 5 minutes and then slowly cooled to room temperature. Then, three times the amount of S-DNA of Cat-1 is added and incubated at 35°C for 15 minutes. In the preparation of Sig-DT, Sig-1, Sig-2 and Sig-3 are first mixed in a 1:1:1 molar ratio, heated and cooled, then three times the amount of T sequence of Sig-1 is added and incubated at 35°C for 15 minutes.

3. The method as described in claim 1 or 2, characterized in that... Step 2) Specifically: The reaction solution contains 10 mM PBS buffer at pH 7.5, containing 100 mM NaCl and 20 mM MgCl2, and the concentrations of Cat-DT and Sig-DT are both 150 nM. The incubation temperature is 37°C.

4. The method as described in claims 1-3, characterized in that... Step 3) Specifically, the fluorescence intensity is measured at 520 nm, and the standard curve is established based on the linear relationship between the logarithm of miR-21 concentration and fluorescence intensity.

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