Beacon probe and application thereof in fluorescence detection of miRNA-21

Abstract

The invention discloses a beacon probe and an application of the beacon probe in fluorescence detection of miRNA-21 (micro Ribonucleic Acid). The probe is prepared from a multifunctional hairpin molecular beacon and a DNA carrier, the DNA carrier is composed of a first probe and a second probe, the sequence of the multifunctional hairpin molecular beacon is shown as SEQ ID NO: 1, the sequence of the first probe is shown as SEQ ID NO: 2, and the sequence of the second probe is shown as SEQ ID NO: 3. The probe provided by the invention is simple in structure, complete in function, low in cost and flexible in design; meanwhile, the provided G-quadruplex cascade sensitization visual detection method of the miRNA-21 has the advantages of being high in sensitivity, high in specificity and the like.

Description

Technical Field

[0001] The present invention relates to a beacon probe and its application in fluorescence detection of miRNA-21 Background Art

[0002] miRNA is a class of single-stranded RNA molecules with a length of 18 to 27 nucleotides, which can regulate the relevant signaling pathways of their target genes, play the role of tumor suppressor genes or oncogenes, and thus affect the occurrence and development of cancer. miRNA-21 is one of the early miRNAs discovered in human cells. Due to the ubiquity of miRNA-21 in various biological processes, people's interest in miRNA-21 has increased dramatically in recent years.

[0003] Existing methods for detecting miRNA-21 include:

[0004] 1. Quantitative real-time fluorescence PCR (qRT-PCR): This method can accurately quantify the expression level of miRNA-21 in biological samples.

[0005] 2. Northern Blotting: Although less used due to its labor-intensive nature, Northern Blotting can provide information on the expression level and molecular weight of miRNA-21.

[0006] 3. Microarray analysis: Microarray is able to detect multiple miRNAs, including miRNA-21, simultaneously, providing a high-throughput screening method in the miRNA-21 expression profile.

[0007] 4. Next-generation sequencing (NGS): NGS technologies, such as RNA-Seq, can identify and quantify the expression levels of miRNA-21, providing insights into the expression profiles of miRNA-21 and other miRNAs in a sample.

[0008] 5. In situ hybridization (ISH): ISH can visualize and localize miRNA-21 in cells or tissues, providing spatial information on its expression pattern.

[0009] 6. Fluorescence in situ hybridization (FISH): Similar to ISH, FISH utilizes fluorescent probes to visualize miRNA-21 within cells.

[0010] However, these methods have their own shortcomings in terms of sensitivity and specificity, cost and throughput, and require professional technicians and instruments. Therefore, it is crucial to develop miRNA-21 detection and analysis methods with high sensitivity, accuracy and wide applicability. Summary of the invention

[0011] The main purpose of the present invention is to provide a branched bimolecular beacon probe reciprocal amplification strategy for enhancing the fluorescence detection method of miRNA-21 to overcome the shortcomings of the prior art.

[0012] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0013] Provided is a branched bimolecular beacon probe for enhancing fluorescence detection of miRNA-21, wherein the probe is made of a multifunctional hairpin molecular beacon and a DNA vector, wherein the DNA vector consists of a first probe and a second probe, the sequence of the multifunctional hairpin molecular beacon is shown in SEQ ID NO:1, the sequence of the first probe is shown in SEQ ID NO:2, and the sequence of the second probe is shown in SEQ ID NO:3.

[0014] A method for preparing the aforementioned branched bimolecular beacon probe for enhancing the fluorescence detection of miRNA-21 is provided, which comprises: reacting a mixture comprising a multifunctional hairpin molecular beacon, a first probe, a second probe and a 10×blue buffer at 95° C. for 5 minutes, cooling at room temperature for more than one hour, to obtain a branched bimolecular beacon probe for enhancing the fluorescence detection of miRNA-21.

[0015] Also provided is the use of the aforementioned branched bimolecular beacon probe for enhancing the fluorescence detection of miRNA-21 in the fluorescence detection of miRNA-21.

[0016] Also provided is a method for enhancing the fluorescence detection of miRNA-21 based on a branched bimolecular beacon probe reciprocal amplification strategy for non-medical diagnosis purposes, which comprises:

[0017] Provided is the aforementioned branched bimolecular beacon probe for enhancing fluorescence detection of miRNA-21;

[0018] A series of standard miRNA-21 solutions of different concentrations were mixed with a mixed system comprising the probe, Klenow fragment polymerase, dNTP and ddH2O, respectively, and incubated to obtain amplified products, and then the amplified products were subjected to fluorescence detection.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention firstly designs and provides a new probe: a multifunctional hairpin molecular beacon (denoted as MMB) and a DNA vector (denoted as DNAholder), wherein the DNA vector is composed of a first probe and a second probe (denoted as S1 and S2), ensuring that MMB and DNAholder have very low background signals, so that the analysis of miRNA-21 can be detected by fluorescence analysis;

[0021] (2) The present invention provides a unique signal cycle amplification method, through the DNA polymerization initiated by the MMB built-in primer, the target miRNA-21 can be stripped to continue to initiate the next cycle reaction, thereby improving the sensitivity and specificity of detecting miRNA-21;

[0022] (3) The probe provided by the present invention can detect two miRNA-21 at one time, so the two fluorescent groups restore fluorescence, which can significantly improve the sensitivity and specificity of detecting miRNA-21. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art description. Obviously, the drawings described below only represent some embodiments of the present invention. For ordinary technicians in this field, other drawings can still be obtained based on these drawings without creative work.

[0024] Figure 1 This is a schematic diagram of an experiment in a typical implementation scheme of the present invention;

[0025] Figure 2 It is a feasibility verification diagram of a typical implementation scheme of the present invention;

[0026] Figure 3 is a sensitivity measurement diagram in a typical implementation scheme of the present invention;

[0027] Figure 4 is a specificity determination diagram in a typical embodiment of the present invention;

[0028] Figure 5 This is a real-time fluorescence change diagram of the system response to 25nM miRNA-21 based on BBMP (B) and dissociation probe system (C) in a typical embodiment of the present invention. ;

[0029] Figure 6 Graph showing serum stability of BBMP (A), dissociation probe system (B) and MMB (C) in a typical embodiment of the present invention. DETAILED DESCRIPTION

[0030] In view of the defects of the prior art, the technical solution of the present invention is obtained by the inventor of this case through long-term research and extensive practice. The technical solution of the present invention will be described clearly and completely below. Obviously, the embodiments only represent some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0031] Specifically, as one aspect of the technical solution of the present invention, the branched bimolecular beacon probe for enhancing the fluorescence detection of miRNA-21 is involved, and the probe is made of a multifunctional hairpin molecular beacon and a DNA vector, and the DNA vector is composed of a first probe and a second probe. The sequence of the multifunctional hairpin molecular beacon is shown in SEQ ID NO:1, the sequence of the first probe is shown in SEQ ID NO:2, and the sequence of the second probe is shown in SEQ ID NO:3.

[0032] In some preferred embodiments, the multifunctional hairpin molecular beacon has a hairpin structure.

[0033] In some preferred embodiments, the first probe and the second probe are denoted as S1 and S2, respectively.

[0034] The probe of the present invention adopts a combination of MMB, S1 and S2 to enable the probe to have fluorescence reporting ability. The presence of target miRNA-21 can trigger DNA polymerization initiated by the built-in primer of MMB, so that the target miRNA-21 can be stripped to continue to start the next round of cycle reaction, and the fluorescence is restored at the same time.

[0035] Another aspect of an embodiment of the present invention also provides a method for preparing the aforementioned branched bimolecular beacon probe for enhancing the fluorescence detection of miRNA-21, which comprises: reacting a mixture comprising a multifunctional hairpin molecular beacon, a first probe, a second probe and a 10×blue buffer at 95°C for 5 minutes, cooling at room temperature for more than one hour, to obtain a branched bimolecular beacon probe for enhancing the fluorescence detection of miRNA-21.

[0036] In some preferred embodiments, the volume ratio of the multifunctional hairpin molecular beacon, the first probe and the second probe is 2:1:1; the concentrations of the multifunctional hairpin molecular beacon, the first probe and the second probe are 0.1 μM, 0.05 μM and 0.05 μM respectively.

[0037] Another aspect of the embodiments of the present invention further provides the use of the aforementioned branched bimolecular beacon probe for enhancing the fluorescence detection of miRNA-21 in the fluorescence detection of miRNA-21.

[0038] The embodiment of the present invention also provides a method for enhancing the fluorescence detection of miRNA-21 based on a branched bimolecular beacon probe reciprocal amplification strategy for non-medical diagnosis purposes, which includes:

[0039] A series of standard miRNA-21 solutions of different concentrations were mixed with a mixed system comprising the probe, Klenow fragment polymerase, dNTP and ddH2O, respectively, and incubated to obtain amplified products, and then the amplified products were subjected to fluorescence detection.

[0040] In some preferred embodiments, the volume ratio of the probe, Klenow fragment polymerase, dNTPs and ddH2O in the mixed system is 18:0.5:2.5:1.5.

[0041] Furthermore, the concentration of the Klenow fragment polymerase is 5 U / μL.

[0042] Furthermore, the concentration of the dNTPs is 10 mM.

[0043] In some preferred embodiments, the mixed system comprises 0.2 μL of miRNA-21, 1.5 μL of ddH2O, 2.5 μL of 10 mM dNTP, 0.5 μL of 5 U / μL Klenow fragment polymerase and 18 μL of BBMP.

[0044] In some preferred embodiments, the fluorescence detection comprises: diluting 25 μL of the reaction solution with 175 μL of 10×blue buffer for subsequent fluorescence measurement.

[0045] In some preferred embodiments, the branched bimolecular beacon probe reciprocal amplification strategy is used to enhance the fluorescence detection method of miRNA-21, specifically comprising: mixing a series of standard miRNA-21 solutions of different concentrations with a mixed system comprising the probe, Klenow fragment polymerase, dNTPs and ddH2O and incubating at 37°C for 2 hours, then diluting the resulting system with 175 μL 10×blue buffer for subsequent fluorescence measurement.

[0046] In some more specific embodiments, the method for enhancing the fluorescence detection of miRNA-21 based on the branched bimolecular beacon probe reciprocal amplification strategy comprises:

[0047] (1) PAGE gel electrophoresis analysis

[0048] Samples for gel electrophoresis analysis were prepared by mixing 8 μL of the resulting reaction mixture, 2 μL of 6× DNA loading buffer, and 2 μL SYBR Green I. 8% nPAGE was prepared on a GelDoc Go imaging system (Bio-Rad, USA) and run at a constant voltage of 90 V using 0.5× TBE buffer as the working buffer.

[0049] (2) Fluorescence detection of miRNA-21

[0050] Prior to the assay, 0.1 μM MMB (2 μL), 0.05 μM S1 (1 μL), and 0.05 μM S2 (1 μL) were added to 1.0 μL of 10× blue buffer (100 mM Tris-HCl, 500 mM NaCl, 100 mM MgCl2, 10 mM DTT) to construct the BBMP system. The mixture was diluted to 10 μL and mixed thoroughly, then heated at 95°C for 5 min and then slowly cooled to room temperature for 1 h to form the BBMP. 0.2 μL of miRNA-21, 1.5 μL of ddH2O, 2.5 μL of 10 mM dNTPs, 0.5 μL of 5 U / μL Klenow fragment polymerase, and 18 μL of BBMP were added. The mixture was incubated at 37°C for 2 h, and then the resulting system was diluted with 175 μL of 10× blue buffer to a final volume of 200 μL for fluorescence measurement. Fluorescence was scanned on a Hitachi F-7100 fluorescence spectrometer and the fluorescence emission spectra were recorded from 500 to 600 nm. The fluorescence intensity at 522 nm was used for quantitative analysis.

[0051] The technical solution of the present invention is further described in detail below in conjunction with a plurality of preferred embodiments and the accompanying drawings. This embodiment provides a detailed implementation method and a specific operation process based on the technical solution of the invention. However, the protection scope of the present invention is not limited to the embodiments described below.

[0052] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0053] The oligonucleotides used (see Table S1) were synthesized by HPLC purification by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). All DNA sequences were diluted to a stock concentration of 10 μM with 1×TE buffer (89 mM Tris-boric acid, 2 mM EDTA, pH 8.3, Beyotime Biotechnology, Beijing) and stored at 4°C. Klenow fragment polymerase (5000 U / mL) and NEBuffer 2 were purchased from Novizan Biotechnology Co., Ltd. (Nanjing, China). Deoxyribonucleotide triphosphate (dNTPs) mixture was provided by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Low molecular weight DNA Maker was obtained from New England Biolabs, Inc. (Beijing, China). SYBR Green Ι was provided by Solebao Biotechnology Co., Ltd. (Beijing, China). Phosphate buffer (10×PBS) was provided by Thermo Fisher Scientific (Hyclone). All reagents were of analytical grade, and solutions were prepared with ultrapure water obtained from MiNiQ-Drect 8 (resistance >18.25 MΩ.cm).

[0054] Instrumentation

[0055] Fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrometer equipped with a xenon lamp used as the excitation source (Hitachi, Ltd., Japan). Gel electrophoresis was performed on a Bio-rad ChemDoc XRS (Bio-Rad, USA), and gels were imaged by the GelDocGo imaging system (Bio-Rad, USA). All experimental procedures were performed according to the manufacturer's protocol.

[0056] The sequences used in the present invention are shown in the following table:

[0057]

[0058]

[0059] Example 1

[0060] Feasibility verification

[0061] (1) Preparation of probe: 0.1 μM MMB (2 μL), 0.05 μM S1 (1 μL), and 0.05 μM S2 (1 μL) were added to 1.0 μL of 10× blue buffer (100 mM Tris-HCl, 500 mM NaCl, 100 mM MgCl 2, 10 mM DTT) for the construction of BBMP system. The mixture was diluted to 10 μL and mixed well, then heated at 95 °C for 5 min and then slowly cooled to room temperature for 1 h to form BBMP.

[0062] (2) Repeat the previous steps to prepare several solutions.

[0063] (3) Fluorescence detection of miRNA-21: 0.2 μL of miRNA-21, 1.5 μL of ddH2O, 2.5 μL of 10 mM dNTP, 0.5 μL of 5 U / μL Klenow fragment polymerase and 18 μL of BBMP were incubated at 37°C for 2 h. Subsequently, the resulting system was diluted with 175 μL of 10× blue buffer to a final volume of 200 μL for fluorescence measurement. Fluorescence was scanned on a Hitachi F-7100 fluorescence spectrometer, and the fluorescence emission spectrum from 500 to 600 nm was recorded. The fluorescence intensity at 522 nm was used for quantitative analysis. The experimental results are shown in Figure 2. Figure 2 As shown, when miRNA-21 was absent, there was only a lower fluorescence intensity, while when miRNA-21 was present, although the fluorescence intensity of BBMP was enhanced, a stronger fluorescence intensity was produced when the two acted together.

[0064] Example 2

[0065] Sensitivity determination

[0066] (1) A series of miRNA-21 samples ranging from 0 to 60 nM were prepared

[0067] (2) In step (3) of Example 1, different concentrations of miRNA-21 samples were added, and fluorescence measurement was performed after the reaction. The measurement results were as follows: Figure 3 As shown, the regression equation can be calculated as F = 3.96C + 18.54, and the correlation coefficient is R 2 =0.9994. F and C represent the fluorescence intensity and miRNA-21 concentration, respectively.

[0068] Example 3

[0069] Specificity determination

[0070] (1) Prepare blank control, single-base mismatch targets (MT1-6 and MT1-7), double-base mismatch targets (MT2-7 and MT2-8), triple-base mismatch targets (MT3-1 and MT3-8), and liquids with the same concentration of miR-155, miR-141, miR-31, miR-122, miRNA-21a, let-7a, and miRNA-21.

[0071] (2) In step (3) of Example 1, the liquid prepared in step (1) was added respectively, and fluorescence measurement was performed after the reaction. The measurement results were as follows: Figure 4 As shown in the figure, only miRNA-21 produced the highest relative fluorescence intensity, while the mismatched bases of MT1, MT2 and MT3 increased and the absorbance signal decreased accordingly. Compared with the blank control, miR-155, miR-141, miR-31, miR-122, miRNA-21a and let-7a could hardly induce any fluorescence intensity.

[0072] Comparative Example 1

[0073] The four solutions containing S1+S2+MMB+KF, S1+S2+MMB+KF+miRNA-21, S1+MMB+KF and S1+MMB+KF+miRNA-21 were reacted and then fluorescence was measured. The results are as follows: Figure 5 As shown, in the presence of target miRNA, a significant fluorescence signal was observed, while the dissociation probe system exhibited a rather weak response, which confirmed that the reaction kinetics of the BBMP system was increased by the steric confinement effect based on the DNA vector.

[0074] Comparative Example 2

[0075] In order to study the biostability of DNA probes against nuclease degradation, a buffer solution containing fetal bovine serum (FBS, 10% v / v) was used to simulate physiological conditions. Degradation assays were then performed by incubating the BBMP system with fetal bovine serum (FBS, 10% v / v) solution for different times. The dissociation probe system and hairpin-shaped molecular beacons were designed as controls. Figure 6 As shown in A, BBMP channels can be observed in 10% FBS within 24 hours. In sharp contrast, the dissociation probe system ( Figure 6 B) and hairpin-shaped molecular beacons ( Figure 6 The lane in C) disappears after 4 hours of exposure to 10% FBS. Comparison of gel images shows that BBMP exhibits higher stability due to the branched DNA nanostructure. Quantitative analysis of the gel bands shows that about 80% of the BBMP can remain intact after 1.5 hours of incubation, which is sufficient for the system to complete the detection within the optimal response time.

[0076] It should be understood that the technical solution of the present invention is not limited to the scope of the above specific embodiments. As long as it complies with the purpose and claims of the present invention, any technical variation based on the technical solution belongs to the protection scope of the present invention.

Claims

1. A beacon probe, characterized in that: The probe is made of a multifunctional hairpin molecular beacon and a DNA vector, wherein the DNA vector is composed of a first probe and a second probe, the sequence of the multifunctional hairpin molecular beacon is shown in SEQ ID NO:1, the sequence of the first probe is shown in SEQ ID NO:2, and the sequence of the second probe is shown in SEQ ID NO:

3.

2. A beacon probe as claimed in claim 1, characterized in that: include: A mixture containing a multifunctional hairpin molecular beacon, a first probe, a second probe and a 10×blue buffer was reacted at 95° C. for 5 minutes and cooled at room temperature for more than one hour to prepare a branched bimolecular beacon probe for enhancing the fluorescence detection of miRNA-21.

3. A beacon probe as claimed in claim 1, characterized in that: The volume ratio of the multifunctional hairpin molecular beacon, the first probe and the second probe is 2:1:1; the concentrations of the multifunctional hairpin molecular beacon, the first probe and the second probe are 0.1 μM, 0.05 μM and 0.05 μM respectively.

4. Application of a beacon probe in fluorescence detection of miRNA-21, characterized in that: Used in fluorescence detection.

5. The use of a beacon probe according to claim 4 in the fluorescence detection of miRNA-21, characterized in that: The volume ratio of the probe, Klenow fragment polymerase, dNTPs and ddH2O in the mixed system is 18:0.5:2.5:1.5; preferably, the concentration of the Klenow fragment polymerase is 5U / μL; preferably, the concentration of the dNTPs is 10mM.

6. Use of a beacon probe according to claim 4 in the fluorescence detection of miRNA-21, characterized in that: A series of standard miRNA-21 solutions of different concentrations were mixed with the mixed system containing the probe, Klenow fragment polymerase, dNTPs and ddH2O and incubated at 37°C for 2 h. Subsequently, the obtained system was diluted with 175 μL 10× blue buffer for subsequent fluorescence measurement.

7. Use of a beacon probe according to claim 4 in the fluorescence detection of miRNA-21, characterized in that: The test sample containing miRNA-21 was mixed with a mixture system comprising the probe, Klenow fragment polymerase, dNTPs and ddH2O and incubated at 37°C for 2 h. Subsequently, the resulting system was diluted with 175 μL 10× blue buffer for subsequent fluorescence measurement.

8. A fluorescence detection method for non-medical diagnosis purposes, characterized in that: include: Providing a beacon probe as claimed in claim 1; A series of standard miRNA-21 solutions of different concentrations were mixed with a mixed system comprising the probe, Klenow fragment polymerase, dNTP and ddH2O, respectively, and incubated to obtain amplified products, and then the amplified products were subjected to fluorescence detection.

Images