Method for miRNA-21 detection by APE1-mediated cascade signal amplification strategy

Abstract

The invention discloses a method for detecting miRNA-21 by an APE1-mediated cascade signal amplification strategy, which comprises the following steps: hybridizing H1 and RC in advance to form a hairpin structure with partial double strands, then mixing the H1-RC hairpin structure, hairpin structure H2, APE 1 enzyme, RP, NEBuffer TM4 and cell lysate for reaction, and finally measuring the fluorescence intensity under the excitation of 485 nm by a microplate reader, so as to determine the miRNA-21 content in the H1-RC hairpin structure, the H1-RC hairpin structure, the H1-RC hairpin structure, the APE 1 enzyme, the RP, the NEBuffer TM4 and the cell lysate. The existence of the miRNA-21 in the cell lysate is judged according to the intensity of the generated fluorescence signal. According to the method, miRNA-21 target determination as low as fM can be realized, the complementary sequences of H1 and H2 are located in the stem and cannot be spontaneously hybridized, and H1 can expose the complementary sequences in the stem only when the target exists, so that CHA reaction is triggered, and the specificity and accuracy of miRNA-21 analysis are ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection, and in particular relates to a method for using an APE1-mediated cascade signal amplification strategy for miRNA-21 detection. Background Art

[0002] Studies have shown that changes in miRNA types and their expression levels are closely related to the progression of various tumor diseases. Therefore, miRNA is expected to be used for early cancer diagnosis and as a biomarker for screening, prognosis and metastasis detection. However, due to its low abundance, miRNA amplification is necessary to improve the sensitivity of its detection. At present, there are corresponding miRNA detection methods, but there are problems such as low sensitivity or complex methods.

[0003] Currently, the detection of miRNAs is mainly accomplished through two strategies: first, by amplifying the miRNA target to increase its concentration - methods include polymerase chain reaction (PCR), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) and exponential isothermal amplification; second, by utilizing the target in specific reactions, such as enzyme-assisted reactions, hybridization chain reactions (HCR), catalytic hairpin assembly (CHA), CRISPR technology and DNA enzyme-assisted reactions to enhance the detection signal. Enzyme-free DNA nanodevices have attracted widespread attention due to their biocompatibility, predictability and programmability, and have been widely used in the field of biosensors. Among them, CHA has been effectively used to detect a variety of analytes, including DNA, mismatched DNA, miRNA, proteins and metal ions. The miRNA-triggered catalytic hairpin assembly reaction enables signal amplification without the involvement of enzymes, thereby simplifying the miRNA detection workflow and reducing costs. However, relying on CHA alone is not sufficient for sensitive detection of miRNAs. To improve the sensitivity and specificity of this method, several cascade signal amplification strategies have been developed by combining CHA with other technologies, such as DNA enzyme-assisted amplification and CRISPR / Cas systems. However, the limited catalytic efficiency of DNAzymes restricts rapid and sensitive miRNA analysis, while the requirement of sgRNAs in CRISPR / Cas increases cost and complexity. Therefore, the development of new strategies for simple, rapid and sensitive CHA-based miRNA detection remains a challenge. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a method for detecting miRNA-21 using an APE1-mediated cascade signal amplification strategy.

[0005] The specific technical solutions are as follows: A method for detecting miRNA-21 using an APE1-mediated cascade signal amplification strategy comprises the following steps: 1) Formation of H1 and RC: NEBuffer TM 2. The hairpin structure H1 shown in SEQ ID NO.1 and the single-stranded RC shown in SEQ ID NO.2 are mixed into a 20 μL system, and H1 and RC are hybridized to form a partially double-stranded hairpin structure through annealing reaction; 2) CHA amplification reaction: In the reaction, the H1-RC hairpin structure prepared in step 1), the hairpin structure H2 with a sequence number as shown in SEQ ID NO.3, APE 1 enzyme, RP with a sequence of 5'-FAM-GCGTCGCG-BHQ1-3', and NEBuffer TM 4 and cell lysate were mixed into a 20 μL system, and the reaction mixture was placed in a 37 °C water bath for reaction; 3) After the reaction in step (2) is completed, the resulting solution is transferred to a black 384-well microplate, and the fluorescence intensity is measured by an ELISA reader under 485 nm excitation. The presence of miRNA-21 with a sequence number such as SEQ ID NO. 4 in the cell lysate is determined by the intensity of the generated fluorescence signal.

[0006] Furthermore, the annealing process of step 1) is 95°C, 1 min; 80°C, 1 min; 70°C, 1 min; 60°C, 1 min; 50°C, 1 min; 40°C, 1 min; 30°C, 1 min; 20°C, 1 min.

[0007] Furthermore, the reaction time in step 2) is 60-90 min.

[0008] Furthermore, in step 2), the amount of APE 1 enzyme added is 50-100 nM.

[0009] Further, after mixing, the molar concentration ratio of APE 1 enzyme to H1-RC hairpin structure is 1:0.8-1.

[0010] Furthermore, the molar concentration ratio of the H1 hairpin structure to the H2 hairpin structure is 1-2:1.

[0011] The beneficial effects of the present invention are: This method can achieve miRNA-21 target determination as low as femtomolar (fM) levels. The complementary sequences of H1 and H2 are located in the stem and cannot hybridize spontaneously. Only when the target is present can H1 expose the complementary sequence in the stem, thereby triggering the CHA reaction, ensuring the specificity and accuracy of miRNA-21 analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the reaction principle diagram of the present invention; Figure 2 This is the optimization result diagram of the reaction system; Figure 3 This is the sensitivity test result diagram; Figure 4 It is the specificity test result diagram; Figure 5 This is the accuracy test result diagram. DETAILED DESCRIPTION

[0013] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0014] like Figure 1 As shown in the figure, a cascade catalytic network is composed based on the catalytic hairpin assembly system (CHA) and the APE1 enzyme. The scheme is divided into two modules: an allosteric module, which is responsible for recognizing specific targets to participate in the CHA cycle reaction, and a signal amplification module, which is used to output fluorescent signals. The allosteric module mainly uses two rationally designed hairpins H1 and H2 to participate in the reaction. The designed H1 mainly includes a target recognition region and a RC sequence complementary region. Through the annealing reaction, H1 hybridizes with the RC sequence to form a partially double-stranded hairpin structure. When there is no target miRNA, H1-RC remains stable and no CHA reaction occurs. However, when the target miRNA-21 is present, the target miRNA-21 binds to the exposed foothold domain a* of H1, triggering branch migration and generating a miRNA-H1-RC intermediate through domain hybridization. The exposed foothold domain in the miRNA-H1-RC intermediate complex further triggers binding to the H2 domain b*, and then the target miRNA is released to start a new cycle. The released RC sequence binds to the RP sequence to form a double-stranded structure and enters the signal amplification module. By modifying the AP site at the appropriate position of RP, FAM and BHQ 1 were modified at the 5' and 3' ends of RP, respectively. APE1 can effectively recognize the RC / RP double strand containing the AP site and remove the AP site in RP, causing RP to break into two parts, releasing the fluorescent probe, and thus generating a high fluorescence signal. Since the concentration of the target is related to the fluorescence intensity, high-sensitivity quantification of miRNA-21 is achieved by measuring the fluorescence intensity of FAM. In addition, because the complementary sequences of H1 and H2 are located in the stem and cannot hybridize spontaneously, only when the target is present can H1 expose the complementary sequence in the stem, thereby triggering the CHA reaction, ensuring the specificity of miRNA-21 analysis.

[0015] Example 1 1. Operation process (1) Formation of H1 and RC Add 2 μL of 10× NEBuffer TM2. 2 μL of 10 μM hairpin structure H1 with sequence number SEQ ID NO.1 and 2 μL of 10 μM single-stranded RC with sequence number SEQ ID NO.2 were added with DEPC water to make up to 20 μL system. Through annealing reaction, H1 and RC sequences were hybridized to form a partially double-stranded hairpin structure with a final concentration of 1 μM. Annealing conditions were 95℃, 1min; 80℃, 1min; 70℃, 1min; 60℃, 1min; 50℃, 1min; 40℃, 1min; 30℃, 1min; 20℃, 1min.

[0016] (2) CHA amplification reaction In the reaction, 2 μL of H1-RC prepared in step (1) above was mixed with 2 μL of the hairpin structure H2 shown in SEQ ID NO.3, 50 nM APE 1 enzyme, 500 nM RP with the sequence of 5'-FAM-GCGTCGCG-BHQ1-3', and 1 × NEBuffer. TM 4 Mix with miRNA and add sterile water to make up to 20 ul system, and place the reaction mixture in a 37 ℃ water bath for 1 hour.

[0017] (3) Measurement of fluorescence signal After the reaction in step (2) is completed, the resulting solution is transferred to a black 384-well microplate, and the fluorescence intensity is measured by an ELISA reader under 485 nm excitation. The presence of miRNA-21 with a sequence number as shown in SEQ ID NO. 4 in the cell lysate is determined by the intensity of the generated fluorescence signal. The RP fluorescent probe is modified with a fluorescent group FAM at one end, a quenching group BHQ1 at the other end, and an idsp in the middle. When the probe is intact, the quenching group can quench the fluorescent group, but when the RC single chain combines with the RP chain to form a double-stranded structure, the APE 1 enzyme recognizes the idsp site and cuts it, so that the fluorescent group and the quenching group are separated, and the fluorescence is restored.

[0018] All the above concentrations refer to the concentration of the substance in the overall system. 2. Optimization of reaction system Change the reaction time of step 2) to 30min, 45min, 60min, 75min, and 90min respectively. The other operating conditions are the same as the above operation process. The results are as follows Figure 2 As shown in A, the fluorescence value gradually increased in the range of 30-60 min, reached a platform at 60 min and began to level off, indicating that the reaction efficiency of the 60 min system was the highest.

[0019] The dosage of APE 1 in step 2) was changed to 10nM, 30nM, 50nM, 70nM, and 100nM respectively. The other operating conditions were the same as the above operation process. The results are as follows Figure 2 As shown in B, the optimal dosage condition is 50 nM.

[0020] The ratio of APE 1 enzyme to H1-RC was changed to 1:0.2, 1:0.4, 1:0.6, 1:0.8, and 1:1, respectively. The other operating conditions were the same as the above operation process. The results are shown in Figure 2 As shown in Figure C, the reaction efficiency gradually increases with the increase in the amount of H1-RC.

[0021] The dosage ratio of H1 and H2 was changed to 0.5:1, 1:1, 1.5:1, and 2:1 respectively. The other operating conditions were the same as the above operation process. The results are as follows: Figure 2 As shown in D in the figure, the effects presented are different due to different proportions.

[0022] Example 2 Sensitivity test In order to study the sensitivity of this method, the effect of different concentrations of miRNA on fluorescence intensity was tested under optimized experimental conditions (other operating conditions were the same as the operating process of Example 1). Figure 3 As shown, as the concentration of miRNA target increases from 100fm to 10 nM, the fluorescence intensity gradually increases, and targets as low as 100 fM can be distinguished from the background. In the range of 100 fm-10nM, there is a good linear relationship between the fluorescence intensity and the logarithm of the miRNA target concentration. The linear regression equation is F=1.535lg[miRNA-21]-0.96, and the correlation coefficient is R2=0.9830. According to the rule of 3 times the standard deviation of the blank response, the limit of detection (LOD) is calculated to be 34.45 fM. These results show that the biosensor is capable of high-sensitivity detection of miRNA-21.

[0023] Example 3 Specificity test Due to the high homology of miRNA sequences (only 1-2 nucleotide differences), the specificity of this method is very important for miRNA detection. Different interference sequences (other operating conditions are the same as the operating process in Example 1) were selected, including miR-141, miR-122, Let-7a and Let-7b, to explore the specificity of the method (Blank is the blank control group of DEPC water). Figure 4 As shown, the fluorescence intensity of miRNA-21 was higher than that of other control groups, and the fluorescence value of the control group did not change much compared with the background control group, indicating that this method has good specificity.

[0024] Example 4 Accuracy test In order to evaluate whether the proposed method can be used to detect target miRNAs in complex biological samples, the concentration of miRNA-21 in cell lysates from MCF-7 cells, HeLa cells, and HEK 293 T cells was analyzed using the method of the present invention and a commercial RT-qPCR kit (other operating conditions were the same as those in Example 1). Figure 5 As shown, the concentration of miRNA-21 determined by the method of the present invention is in good agreement with the RT-qPCR results. This indicates that the method of the present invention is comparable to the reported method. The results show that the method is applicable to complex biological samples and has good accuracy.

Claims

1. A method for detecting miRNA-21 using an APE1-mediated cascade signal amplification strategy, characterized in that: The steps include: 1) Formation of H1 and RC: NEBuffer TM 2. The hairpin structure H1 shown in SEQ ID NO.1 and the single-stranded RC shown in SEQ ID NO.2 are mixed into a 20 μL system, and H1 and RC are hybridized to form a partially double-stranded hairpin structure through annealing reaction; 2) CHA amplification reaction: In the reaction, the H1-RC hairpin structure prepared in step 1), the hairpin structure H2 with a sequence number as shown in SEQ ID NO.3, APE 1 enzyme, RP with a sequence of 5'-FAM-GCGTCGCG-BHQ1-3', and NEBuffer TM 4 and cell lysate were mixed into a 20 μL system, and the reaction mixture was placed in a 37 °C water bath for reaction; 3) After the reaction in step (2) is completed, the resulting solution is transferred to a black 384-well microplate, and the fluorescence intensity is measured by an ELISA reader under 485 nm excitation. The presence of miRNA-21 with a sequence number such as SEQ ID NO. 4 in the cell lysate is determined by the intensity of the generated fluorescence signal.

2. The method for detecting miRNA-21 using an APE1-mediated cascade signal amplification strategy as claimed in claim 1, characterized in that: The annealing process of step 1) is 95°C, 1 min; 80°C, 1 min; 70°C, 1 min; 60°C, 1 min; 50°C, 1 min; 40°C, 1 min; 30°C, 1 min; 20°C, 1 min.

3. The method for detecting miRNA-21 using an APE1-mediated cascade signal amplification strategy as claimed in claim 1, characterized in that: The reaction time in step 2) is 60-90 min.

4. The method for detecting miRNA-21 using an APE1-mediated cascade signal amplification strategy as claimed in claim 1, characterized in that: In step 2), the amount of APE 1 enzyme added is 50-100 nM.

5. The method for detecting miRNA-21 using an APE1-mediated cascade signal amplification strategy as claimed in claim 1, characterized in that: After mixing, the molar concentration ratio of APE 1 enzyme to H1-RC hairpin structure was 1:0.8-1.

6. The method for detecting miRNA-21 using an APE1-mediated cascade signal amplification strategy as claimed in claim 1, characterized in that: The molar concentration ratio of the H1 hairpin structure to the H2 hairpin structure is 1-2:1.

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