Method for detecting miR-21 based on self-driven and self-catalyzed three-legged DNA nano machine

Abstract

The invention develops a self-driving and self-catalyzing method for detecting microRNA-21 (miR-21) on the basis of a three-legged DNA (deoxyribonucleic acid) nano machine. If a sample to be detected contains miR-21, the miR-21 can open an arm 1 of the three-legged DNA nano machine through a proximity effect, and the arm 1 and a nearby arm 2 form a DNA enzyme structure. The DNA enzyme can cut a substrate chain on the arm 2, the DNA arm 2 exposed after cutting is dissociated to compete with the arm 3, and a DNA fragment (sequence Q) marked with a quenching group is generated to leave the DNA nano-machine, so that a fluorescence signal is recovered. Meanwhile, the released sequence Q can catalyze the next self-driven and self-catalyzed circular reaction of the three-legged DNA nano machine. The component with close space positioning is constructed on a single DNA nanostructure, so that the local reactant concentration and the reaction rate are remarkably improved. And the fluorescence signal and the miR-21 have a linear relationship between the concentration of 10 pM and 50 nM. Compared with a free diffusion reactant, the effective concentration of the reactant is greatly increased. Therefore, the incubation time is obviously shortened to 35 minutes. The strategy has important significance in the aspect of providing reference value for miRNA detection and disease diagnosis.

Description

Technical Field

[0001] The present invention relates to a method for detecting miR-21 based on the self-driving and self-catalytic nature of a tripod DNA nanomachine. Background Art

[0002] MicroRNA (miRNA) is a class of small noncoding RNAs containing 21 to 23 nucleotides that are involved in the regulation of gene expression. Most studies have shown that miRNA levels are altered in cancer patients, indicating their important role in the carcinogenic process. Based on this evidence, miRNA has been regarded as a sensitive and specific biomarker for early detection of cancer. Changes in the levels of miRNA-21 (miR-21) in the serum of patients with Mycoplasma pneumoniae pneumonia (MPP) are significantly related to inflammatory factors and T lymphocyte subsets. The expression level of microRNA miR-21 in serum has an important reference value for the diagnosis of patients with severe pneumonia. At present, common miRNA detection methods RT-qPCR, Northern Blot and microarray are widely used to detect miRNA. The improvement of sensitivity is conducive to the detection of miRNA signals. However, some obvious disadvantages hinder their use, such as long processing time, cumbersome operation process and high false positive results. Therefore, new strategies to simplify the miRNA detection process and shorten the detection time are worth exploring.

[0003] DNA nanomachines are various nanoscale devices and nanostructures with DNA structures as basic components, showing potential performance in drug delivery and biosensing analysis. It has attracted increasing attention due to its good biocompatibility, predictable secondary structure, and good thermal stability and functional modification. They are usually driven by the free energy of base pairing. So far, various DNA nanomachines have been developed, including DNA tweezers, DNA walkers, DNA motors, DNA gears, and DNA nanocages, for biosensor design, drug delivery, and logic molecular computing. Despite significant progress, many existing methods are still challenged due to limited reaction rates. These reaction rates usually reach saturation with the extension of reaction time, mainly due to the presence of diffusible reactants or fuel molecules. In addition, low intracellular DNA concentrations limited by internalization efficiency and biocompatibility may reduce the reaction kinetics in living cells. Therefore, it is highly desirable to develop a DNA nanomachine with high efficiency and high sensitivity for self-driving and self-catalysis.

[0004] In order to solve the problems in the prior art, the present invention proposes a self-driving and self-catalytic detection method for miR-21 detection based on a tripod DNA nanomachine. Summary of the invention

[0005] To solve the problems in the prior art, the present invention proposes a novel method for rapid and sensitive detection of miR-21 based on the self-driving and self-catalytic reaction of a tripod DNA nanomachine. If there is miR-21 in the sample to be tested, miR-21 can open the arm 1 end of the DNA nanomachine. Due to the proximity effect, the released chain combines with the adjacent arm 2 to form a DNA enzyme structure. After the cleavage reaction, the exposed DNA arm 2 region hybridizes with the nearby arm 3, resulting in the departure of the DNA fragment (sequence Q) labeled with a quenching group, thereby restoring the fluorescence intensity. Then, the DNA fragment (sequence Q) with a catalytic effect combines with another DNA nanomachine to start the next self-driving and self-catalytic cycle. There is a dynamic correlation between the fluorescence intensity and the target concentration. Therefore, the concentration of miR-21 can be quantitatively detected by the fluorescence intensity.

[0006] Specifically, the present invention provides a method for detecting miR-21 based on the self-driving and self-catalytic nature of a tripod DNA nanomachine, comprising the following steps:

[0007] 1) Sequences H1, H2 and H3 were dissolved in Tris-HCl buffer at the same molar ratio to form a tripod DNA framework, and then sequence Q was added to the tripod DNA framework solution to form the entire DNA nanomachine structure. The sequence of H1 is 5'-TCAACATCAGTCTGATAAGCTAATCGTTTTGCGGCACTTTGACCGCATAAGCTA-FAM-3', the sequence of H2 is 5'-ATCGATCGATCGTGATGCTGATCGAATrAGGATGCGTTTTTCATAA AACAGTGCCGCTTTTTTTTTTTTTTTTACGCATCAGCGATTAACCAGGTTACACCCATGT TTCGATTAGCTTATC-3', the sequence of H3 is 5'-TCGATCAGCATCAGTCTGGCGGTCAAGTTTTAT GTTTTTTTTTTTTTTTTTTTTTAGCTTATCAGACCGATCGATCGAT-3', and the sequence of Q is 5'-BHQ1-TAGCTTATCAGACTGATGCTGA-3',

[0008] 2) The DNA nanomachine and miR-21 were mixed in a Tris-HCl buffer solution, wherein the sequence of miR-21 was 5'-UAGCUUAUCAGACUGAUGUUGA-3'.

[0009] 3) Detecting the fluorescence spectrum of the solution obtained in step 2), and using the standard curve method to obtain the concentration of miR-21 in the sample to be tested.

[0010] Preferably, step 1) is specifically as follows: dissolving equimolar amounts of sequences H1, H2 and H3 in a Tris-HCl buffer solution (10 mM Tris-HCl, 10 mM MgCl2, pH 7.4), and then incubating at 37°C for 60 minutes to form a tripodal DNA framework. Then, sequence Q is added to the above solution to form the entire DNA nanomachine structure.

[0011] Preferably, step 2) is specifically as follows: a total of 250 μL of Tris-HCl buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl and 10 mM MgCl2) is used to mix 150 nM DNA nanomachine and miR-21. Then, the mixed solution is reacted at 37° C. for 35 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 (A) The formation process of the tripod DNA nanomachine (B) is a schematic diagram of the detection process of the present invention.

[0013] Figure 2 (A) Fluorescence stability of the tripod DNA nanomachine in the presence and absence of miR-21. (B) Fluorescence intensity of different combination samples: (1) H1+H2+H3+Q+Mg 2+ ; (2) H1+H2+H3+Mg 2+ +miR-21; (3) H1+H2+H3+Q+miR-21; (4) H1*+H2*+H3*+Q* (because the sequences of Q* and Q complete different DNA sequences, H1*+H2*+H3* changes the sequence according to Q*) +miR-21+Mg 2+ ; (5) H1+H2+H3+Q+miR-21+Mg 2+ .

[0014] Figure 3 Comparison of the results of the method's selectivity study on miR-21 and other similar sequences. The five samples consisted of the following: blank, miR-16, miR-92a, miR-146a, miR-21, and mixed samples containing miR-21 and other miRNA sequences. Specific embodiments

[0015] The present invention will be further described in detail below in conjunction with the embodiments.

[0016] The detection method and steps of the present invention are as follows:

[0017] 1) Equimolar amounts of sequences H1, H2, and H3 were dissolved in a Tris-HCl buffer solution (10 mM Tris-HCl, 10 mM MgCl2, pH 7.4) and then incubated at 37°C for 60 minutes to form a tripodal DNA framework. Then, sequence Q was added to the above solution to form the entire DNA nanomachine structure. The H1 sequence is 5'-TCAACATCAGT CTGATAAGCTAATCGTTTTGCGGCACTTTGACCGCATAAGCTA-FAM-3', and the H2 sequence is 5'-ATCGATCGATCGTGATGCTGATCGAATrAGGATGCGTTTTTCATAAAACAGTGCCGCTTTT TTTTTTTTTTTACGCATCAGCGATTAACCAGGTTACACCCATGTTTCGATTAGCTTATC-3', H3 sequence is 5'-TCGATCAGCATCAGTCTGGCGGTCAAGTTTTATGTTTTTTTTTTTTTTTTTTTAGCTTATCAGACCGATCGATCGAT-3', Q sequence is 5'-BHQ1-TAGCTTATCAGACTG ATGCTGA-3',

[0018] 2) A total of 250 μL of Tris-HCl buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, and 10 mM MgCl2) was used to mix 150 nM DNA nanomachine and miR-21. The mixed solution was then reacted at 37°C for 35 minutes. The miR-21 sequence is 5'-UAGCUUAUCAGACUGAUGUUGA-3'.

[0019] 3) The fluorescence spectrum was excited at 480 nm, and the emission spectrum was scanned at 505-600 nm. The concentration of miR-21 in the sample to be tested was obtained using the standard curve method.

[0020] The above method is the best embodiment of the present invention.

[0021] The miR-21 detection principle of this method is as follows Figure 1 As shown. miR-21 can open the arm 1 end of the DNA nanomachine. Due to the proximity effect, the released chain binds to the adjacent arm 2 to form a DNA enzyme structure. After the cleavage reaction, the exposed DNA arm 2 region hybridizes with the nearby arm 3, resulting in the departure of the DNA fragment (sequence Q), thereby restoring the fluorescence intensity. Then, the DNA fragment (sequence Q) with a catalytic effect binds to another DNA nanomachine to start the next self-driving and autocatalytic cycle. There is a dynamic correlation between the fluorescence intensity and the target concentration. Therefore, the concentration of miR-21 can be quantitatively detected by the fluorescence intensity.

[0022] Feasibility characterization of the method

[0023] The stability of the nanostructure has a crucial impact on its performance. Therefore, the stability of the tripod DNA nanomachine was monitored by fluorescence signals. Figure 2 As shown in A, the fluorescence intensity of the DNA nanomachine without a target is weak, which means that the DNA nanomachine is in the "off" state. In addition, the fluorescence signal is relatively stable and constant within 5 hours, indicating that the DNA nanomachine has strong stability and can maintain its complete structure during the detection process.

[0024] The feasibility of this self-propelled and self-catalytic tripodal DNA nanomachine was investigated by fluorescence signals of samples with different compositions. Figure 2 B. Sample 1 (blank sample) has a low fluorescence signal due to the lack of target miR-21. However, sample 2 without sequence Q shows a strong fluorescence intensity due to the open state of arm 3 of the tripod DNA nanomachine, and this fluorescence intensity is independent of the concentration of miR-21 in the sample and cannot be used for the detection of miR-21. Sample 3 does not contain Mg 2+ The fluorescence intensity of the sample is weak, indicating that the DNA enzyme cutting is inhibited. When the sequence Q is replaced by Q*, which cannot be complementary to the free part of H1 on the tripod DNA nanomachine, the fluorescence signal of sample 4 is relatively strong, indicating that after replacing the sequence Q with the sequence Q* (because the sequence of Q* and Q completes a different DNA sequence, H1*+H2*+H3* changes the sequence accordingly according to Q*), it cannot catalyze the subsequent cycle reaction. In addition, sample 5 shows a strong fluorescence signal containing all components, indicating that the formation of DNA enzyme, the cutting of S-DNA, the departure of DNA fragments (sequence Q) and the catalytic cycle reaction of the nanomachine are carried out as designed. The results are consistent with the detection principle of this strategy.

[0025] The selectivity of this method for miR-21

[0026] The anti-interference and specificity of the miR-16, miR-92a, miR-146a and miR-21 were evaluated. Figure 3 As shown, the proposed nanomachine showed a strong response only to the miR-2 sample and the mixed sample, indicating that only miR-21 can initiate the self-driving and self-catalytic tripod DNA nanomachine to obtain a strong fluorescence signal ( Figure 3 ). These results indicate that the proposed strategy has good anti-interference and specificity.

[0027] Practicality of miR-21 detection

[0028] In order to evaluate the practical application performance of the strategy, the actual analytical ability of the proposed strategy was evaluated by detecting miR-21 in real samples. Different concentrations of miR-21 were added to 10-fold diluted healthy human serum for recovery test. As shown in Table 1, the recovery rate varied from 92.7% to 107.4%, and the RSD varied from 5.4% to 8.9%. The data clearly show that the strategy was successful in detecting miR-21 in actual human serum. It meets the requirements of practical application.

[0029] Table 1. Practical applications of miR-21 detection in human blood samples

[0030]

[0031] “ND”: Not detected.

[0032] In conclusion, a self-driving and self-catalytic tripod DNA nanomachine was developed for the detection of miR-21. The effective concentration of the reactants was greatly improved by the specific tripod DNA nanomachine design. In addition, the incubation time was greatly shortened to 35 min. Due to the self-driving and self-catalytic strategy, the LOD of miR-21 detection was as low as 3 pM. All the results showed that this method has great value in miRNA detection and provides reference for disease diagnosis.

Claims

1. A method for detecting miR-21 based on self-driving and self-catalytic tripod DNA nanomachines. The method comprises the following steps: 1) Sequences H1, H2 and H3 were dissolved in Tris-HCl buffer at the same molar ratio to form a tripod DNA framework, and then sequence Q was added to the tripod DNA framework solution to form the entire DNA nanomachine structure. The sequence of H1 is 5'-TCAACATCAGTCTGATAAGCTAATCGTTTTGCGGCACTTTGACCGCATAAGCTA-FAM-3', the sequence of H2 is 5'-ATCGATCGATCGTGATGCTGATCGAATrAGGATGCGTTTTTCATAA AACAGTGCCGCTTTTTTTTTTTTTTTTACGCATCAGCGATTAACCAGGTTACACCCATGT TTCGATTAGCTTATC-3', the sequence of H3 is 5'-TCGATCAGCATCAGTCTGGCGGTCAAGTTTTAT GTTTTTTTTTTTTTTTTTTTTTAGCTTATCAGACCGATCGATCGAT-3', and the sequence of Q is 5'-BHQ1-TAGCTTATCAGACTGATGCTGA-3', 2) The DNA nanomachine and the sample to be tested are mixed and reacted in a Tris-HCl buffer solution, and the sequence of miR-21 is 5'-UAGCUUAUCAGACUGAUGUUGA-3'. 3) Detecting the fluorescence spectrum of the solution obtained in step 2), and using the standard curve method to obtain the concentration of miR-21 in the sample to be tested.

2. The method according to claim 1, characterized in that Step 1) is specifically as follows: equimolar amounts of sequences H1, H2 and H3 are dissolved in a Tris-HCl buffer solution (10 mM Tris-HCl, 10 mM MgCl2, pH 7.4), and then incubated at 37°C for 60 minutes to form a tripodal DNA framework. Then, sequence Q is added to the above solution to form the entire DNA nanomachine structure.

3. The method according to claim 2, characterized in that Step 2) is specifically as follows: a total of 250 μL of Tris-HCl buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl and 10 mM MgCl2) is used to mix 150 nM DNA nanomachine and the sample to be tested. Then, the mixed solution is reacted at 37° C. for 35 minutes.

4. The method according to claim 3, characterized in that Step 3) is specifically as follows: the fluorescence spectrum is excited at 480 nm, and the emission spectrum is scanned at 505-600 nm. The concentration of miR-21 in the sample to be tested is obtained using the standard curve method.

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