Double-target cascade gating activated CRISPR / Cas12a system for detecting microRNA155 and FEN1

Abstract

The invention relates to a cascade gating activated CRISPR / Cas12a system which is used for synchronous detection of miRNA155 and FEN1, the system comprises recognition assembly and enzyme digestion of two DNA hairpins, and Cas12a is activated by an enzyme digestion product to generate a fluorescence signal. The DNA hairpin 1 comprises a recognition part of miRNA155, a hidden foothold of the DNA hairpin 2 and a part of Cas12a activation chain, and the DNA hairpin 2 comprises a part combined with the hidden foothold of the DNA hairpin 1 and a part of Cas12a activation chain. In the application, two DNA hairpins are combined and assembled under the catalysis of miRNA155 to be called as an FEN1 enzyme cutting substrate, a CRISPR / Cas12a system can be activated in a cascade gating manner, and signals are generated only when the two targets exist, so that the specificity synchronous detection of the two targets is completed.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a fluorescent sensor for cell detection of miRNA155 and FEN1 and a preparation method thereof. Background Art

[0002] Flap endonuclease-1 (FEN1) is an endonuclease that can specifically cleave the single-stranded protruding branch chain at the 5'-end of double-stranded DNA. Its cleavage specificity is based on the unique DNA branch structure and is not sequence-dependent. FEN1 is widely distributed in the nucleus and cytoplasm, has stable cleavage activity, and plays an irreplaceable role in the maturation and base excision repair of Okazaki fragments. Increased expression of FEN1 has been observed in actively proliferating tissues, and upregulation of FEN1 expression has also been observed in tumor cells of breast cancer, lung cancer, and gastric cancer, suggesting the important role of FEN1 in tumorigenesis. Most current FEN1 detection methods cannot detect the expression level of FEN1 in living single cells, but can only detect FEN1 activity in tumor cell lysates. In situ imaging detection methods in cells mostly use FEN1 to cleave reporter probes with fluorescent groups to generate signals, lacking the signal amplification step. How to more sensitively reflect the expression level of FEN1 in single tumor cells remains to be further explored.

[0003] Micro RNA (miRNA) is a type of non-coding small RNA that plays an important role in the genetic regulation of cell growth and development. MiRNA is transcribed in the cell nucleus, and after maturation, it binds to the corresponding mRNA site through base complementary pairing to regulate gene expression. A variety of miRNAs are closely related to the occurrence and development of tumors. Tumor tissues can be distinguished from normal tissues by detecting the expression level of miRNA. Among the various miRNA sequences, miRNA155 is overexpressed in many tumors, and it promotes the occurrence and development of tumors by inhibiting the apoptosis pathway. Its expression is elevated in a variety of solid tumors such as breast cancer, colon cancer, thyroid cancer, and lung cancer, indicating a poor prognosis. Detecting the expression level of miRNA155 in living cells at the single-cell level is of great significance for the differentiation and screening of tumor cells.

[0004] With the application of nanomaterials and DNA encoding technology, significant progress has been made in the in situ detection of miRNA or FEN1 in cells. Intracellular detection of FEN1 can be achieved by loading DNA probes with nanomaterials, which can generate fluorescent signals or trigger subsequent signal amplification reactions after FEN1 cleavage. Since the expression level of miRNA in cells is very low, a series of signal amplification strategies need to be designed for in situ detection of miRNA in cells. Common isothermal amplification methods such as rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) can achieve the detection of trace nucleic acid molecules in cells. Although significant progress has been made in the detection of miRNA or FEN1 alone, most of these methods require cumbersome synthesis and modification of nanomaterials, or require the participation of multiple enzymes, and cannot simultaneously detect miRNA and FEN1 in cells. Among the methods for synchronously detecting two biological molecules in cells, there are methods for synchronously detecting miRNA and telomerase. However, the enzyme activities of telomerase and FEN1 are different, and these methods cannot meet the requirements of synchronous detection of miRNA and FEN1. Summary of the invention

[0005] Based on the above-mentioned technology, we designed a dual-target cascade detection scheme. When miRNA155 and FEN1 are present at the same time, miRNA155 opens DNA hairpin 1, which is then sheared by FEN1 after binding to DNA hairpin 2. After removing the 5`-end branch, a DNA double strand with a PAM sequence is formed, activating Cas12a and generating a significant fluorescent signal output by trans-cutting the reporter probe. This scheme can achieve more specific detection of two biological molecules through the logic gating strategy of miRNA155 and FEN1. The addition of Cas12a ensures stable signal output, which is expected to provide new ideas for distinguishing tumor tissue from normal tissue and exploring the interaction between the two biological molecules.

[0006] Compared with the current miRNA or FEN1 detection methods, the present invention has the following advantages:

[0007] Compared with the method of detecting miRNA or FEN1 outside the cell, this method can realize the target detection in the cell environment without destroying the integrity of the cell, and can provide more comprehensive and intuitive tumor cell information.

[0008] Compared with the method of detecting miRNA or FEN1 separately in cells, this method can achieve simultaneous detection of both. The assembly of DNA probes is initiated by miRNA to form FEN1 cleavage products, and then the signal is amplified by the CRISPR / Cas12a system. The simultaneous detection of miRNA and FEN1 can reflect more comprehensive tumor information and improve the specificity of tumor cell screening. It has certain application value in studying the interaction between miRNA and FEN1. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a schematic diagram of a dual-target cascade-gated activated CRISPR / Cas12a system for detecting miRNA155 and FEN1.

[0010] Figure 2 The electrophoresis diagram of the dual-target cascade-gated activated CRISPR / Cas12a system detecting miRNA155 and FEN1 to verify the feasibility of hairpin assembly and FEN1 cleavage, as well as the fluorescence signal comparison diagram of CRISPR / Cas12a systems with different components are shown in the figure.

[0011] Figure 3 This is a comparison chart of fluorescence signals specific to different types of miRNAs and different types of DNA enzymes detected by a dual-target cascade-gated activated CRISPR / Cas12a system of the present invention.

[0012] the term:

[0013] FEN1: flap endonuclease-1

[0014] miRNA: microRNA

[0015] CRISPR / Cas12a: Clustered regularly interspaced short palindromic repeats / associated protein 12a

[0016] crRNA:CRISPR RNA

[0017] PAM: Protospacer adjacent motif DETAILED DESCRIPTION

[0018] We designed a detection sequence for dual-target cascade activation based on the RNA sequence of miRNA155.

[0019] Schematic diagram Figure 1As shown, when miRNA155 is present, the 3'-end of DNA hairpin 1 (HP1) is complementary to miRNA155, and the combination of the two causes a portion of the hairpin stem of HP1 to unfold, exposing a hidden foothold. The 3'-end of DNA hairpin 2 (HP2) binds to the exposed foothold and completely unfolds the stem loop of HP1. miRNA155 opens HP1, and HP1 then binds to HP2. The complex miRNA155-HP1-HP2 formed by the three sequences constitutes a double-stranded structure with a 5'-end branch. The branch part is a partial stem loop sequence of HP1, and the hybrid part of HP1 and HP2 contains a sequence complementary to crRNA. The composite structure formed by the three sequences can be structurally specifically cleaved by FEN1 to remove the 5'-end branch of the composite structure.

[0020] After FEN1 removes the 5'-end branch of the composite structure, the gap produced by FEN1 shearing is connected by T4 ligase. Without the obstruction of the 5'-end branch, the hybridized part of HP1 and HP2 binds to crRNA, thereby activating the trans-cutting activity of Cas12a. FQ reporter probe is added to the system. The reporter probe is a short DNA chain of 5nt, and the fluorescent group FAM and the quenching group BHQ are modified at both ends. After Cas12a shearing, FAM is away from BHQ and the quenching effect of BHQ is released, thereby generating a fluorescent signal.

[0021] In the above detection system, since it is the part of HP1 and HP2 hybridization that activates Cas12a, separate HP1 and HP2 cannot activate Cas12a, and only the combination of miRNA155 and HP1 can open the subsequent reaction. When miRNA155 is not present in the system, HP1 and HP2 do not bind. Similarly, the complex of miRNA155-HP1-HP2 hinders the complementary recognition with crRNA due to the presence of 5'-end branches, and Cas12a cannot be activated. Such a design can reduce the background signal and ensure the specificity of the detection at the same time. Only when two targets exist at the same time, Cas12a can be activated to produce a significant fluorescent signal, thereby realizing the sensitive detection of miRNA155 and FEN1 in the cell.

[0022] Experimental Materials

[0023] The oligonucleotide chains purified by HPLC and DEPC-treated water were purchased from Sangon Biotechnology (Shanghai) Co., Ltd. T4 DNA ligase and Phi29 DNA polymerase were purchased from Thermo Fisher Scientific (USA), thermostable FEN1, lbaCas12a protein, 10×nebuffer2.0, Bst2.0WarmStart DNA polymerase, APE1 were purchased from New England Biolabs (Beijing) Co., Ltd., 6× loading buffer and 20-200bp DNA ladder were purchased from Takara Biotech (Dalian) Co., Ltd. All chemical reagents were analytical grade and used directly without further purification. All aqueous solutions were prepared using DEPC-treated water.

[0024] Experimental Instruments The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope that the present invention is intended to protect.

[0025] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0026] Experimental instruments

[0027] The electrophoresis instrument used for native polyacrylamide gel electrophoresis (PAGE) was purchased from Bio-Ras (USA), and the fluorescence spectrophotometer F-4700 used for fluorescence spectrum measurement and fluorescence kinetics detection was purchased from Hitachi-hightech (Japan).

[0028] The nucleotide sequences involved in the present invention are shown in Table 1.

[0029] Table 1

[0030]

[0031]

[0032] Implementation Case 1:

[0033] 1.1. Assembly and shearing of cascade gating probes for miRNA155 and FEN1:

[0034] The cascade probe assembly and shearing system of miRNA155 and FEN1 was constructed according to the following steps. All sequences used were dissolved in DEPC-treated water. HP1 and HP2 were annealed in 1× Nebuffer 2.0 before use. The annealing process was heating at 95°C for 5 min, slowly cooling to room temperature, and refrigerating at -20°C for later use.

[0035] Add miRNA155 (1ul 100nM), HP1 (1ul 1000nM), HP2 (1ul 1000nM), FEN1 (0.1U), 10×ThermolPol (1ul) to an enzyme-free EP tube, add DEPC-treated water to 10ul, mix the above system, and incubate at 37°C for 60min.

[0036] 1.2. Ligation with T4 ligase:

[0037] Add 10×T4 ligase buffer (2 ul) and T4 ligase (0.5 U) to the mixed solution in step (1), add DEPC-treated water to 20 ul, mix the above system, and incubate at 37°C for 30 min.

[0038] 1.3. Activation of CRISPR / Cas12a system and fluorescence signal detection:

[0039] Cas12a (1.25ul 1uM) and crRNA (1.25ul 1uM) were mixed and incubated at 37°C for 25min. 2.5ul of the mixture of Cas12a and crRNA, 10×Nebuffer2.0 (5ul), F-Qreporter (2.5ul 10uM) were added to the mixed solution in step (2), DEPC-treated water was added to 50ul, and incubated at 37°C in the dark for 30min to obtain a fluorescence detection system.

[0040] Take 50ul of the above reaction solution and add it to a quartz cuvette, and measure it using a fluorescence spectrophotometer. The specific steps are as follows:

[0041] Fluorescence cuvette cleaning: Soak the cuvette in anhydrous ethanol and then clean it with DEPC-treated water;

[0042] Setting parameters: set the excitation wavelength to 492nm, the detection emission wavelength to 500-600nm, the voltage to 600V, the slit to 5nm, and the scanning rate to 1200nm / min;

[0043] Zero adjustment: Add DEPC-treated water to the cuvette for zero adjustment;

[0044] To perform the test: add the reaction solution into the cuvette and click on the test button to obtain the fluorescence signal value.

[0045] Implementation Case 2

[0046] Verify the feasibility of the testing system

[0047] The overall detection process in implementation case 1 was verified by PAGE electrophoresis, and the results are as follows: Figure 2 As shown in A, the lane marker is a 20bp-500bp DNA marker;

[0048] Lane 1 is HP1;

[0049] Lane 2 is HP2;

[0050] Lane 3, HP1 and HP2;

[0051] Lane 4: HP1, HP2, and FEN1;

[0052] Lane 5: HP1, HP2, and miRNA155;

[0053] Lane 6 is HP1, HP2, miRNA155, and FEN1.

[0054] like Figure 2 As shown in A, lanes 1 and 2 correspond to HP1 and HP2, respectively. The band in lane 3 corresponds to the bands in lanes 1 and 2, and there are no other bands, indicating that HP1 and HP2 are not bound. Lane 4 indicates that HP1 and HP2 are not bound and will not be cut by FEN1. In lane 5, in addition to the bands of HP1 and HP2, there is a new band, which means that miRNA155 opens HP1 and binds to HP2, indicating that the assembly is successful by miRNA155 opening HP1 and then binding to HP2. Lane 6 also has a band produced by the combination of HP1, HP2, and miRNA155, and a new band appears under the shearing action of FEN1, indicating that the above hairpin assembly products can be recognized and cut by FEN1.

[0055] like Figure 2 As shown in B, the spectral curves represent no target (HP1+HP2), single target (HP1+HP2+miRNA155, HP1+HP2+FEN1), and double target (HP1+HP2+miRNA155+FEN1). It can be seen that when only a single target exists, the fluorescence signal is very low, and only when both targets are present, the fluorescence signal will be restored, proving the feasibility of the system.

[0056] Implementation Case 3

[0057] To verify the specificity of the CRISPR / Cas12a system activated by the cascade gated probe of the present invention in detecting miRNA155 and FEN1, we set up a series of control groups to measure the fluorescence signal intensity generated by miRNAs of different sequences and different types of DNA enzymes. Figure 3 As shown, among the combinations of various enzymes and miRNAs, only the combination of miRNA155 and FEN1 produced an obvious fluorescent signal, and other combinations could not produce significant fluorescent signals because miRNA could not open HP1, or could not be sheared to form a double strand that activated Cas12a after miRNA155 opened HP1. In addition, in the mixed miRNA, the detection system of the present invention can resist the interference of other sequences, identify miRNA155, and produce a fluorescent signal under the shearing of FEN1, thereby indicating that a cascade gated probe-activated CRISPR / Cas12a system of the present invention can specifically identify miRNA155 and FEN1.

Claims

1. A dual-target cascade-gated activated CRISPR / Cas12a system detects microRNA155 and FEN1, It is characterized in that The recognition, assembly and enzyme cleavage of the two DNA hairpins activate Cas12a to generate a fluorescent signal through the enzyme cleavage product; DNA hairpin 1 includes a recognition part of miRNA155, a hidden foothold of DNA hairpin 2 and a part of the Cas12a activation chain, and DNA hairpin 2 includes a part binding to the hidden foothold of DNA hairpin 1 and a part of the Cas12a activation chain.

2. The cascade-gated activated CRISPR / Cas12a system according to claim 1, It is characterized in that The nucleotide sequence of the DNA hairpin 1 is shown in Table 1HP1.

3. The cascade gated activated CRISPR / Cas12a system as claimed in claim 1, It is characterized in that The nucleotide sequence of the DNA hairpin 2 is shown in Table 1HP2.

4. The preparation method of the cascade gated activated CRISPR / Cas12a system according to claim 1, characterized in that the preparation method include: The DNAs are annealed separately for later use, T4 ligase is added to the buffer system, the annealed DNA hairpin is added, Cas12a and crRNA are mixed and incubated, and then added to the above reaction system, and the FQ reporter probe is added to obtain the cascade gated activated CRISPR / Cas12a system.

5. The preparation method according to claim 4, It is characterized in that The annealing temperature is 95°C and the annealing time is 5 min.

6. The preparation method according to claim 4, It is characterized in that The incubation temperature was 37°C.

7. A fluorescence detection method, using the cascade-gated activated CRISPR / Cas12a system according to claim 1 and / or the fluorescence system prepared according to claim 4.

8. The fluorescence detection method according to claim 6, It is characterized in that The determination method comprises: soaking the cuvette in anhydrous ethanol, then washing it with DEPC-treated water, setting parameters including excitation wavelength, detection emission wavelength, voltage, slit, scanning rate, etc., adding DEPC-treated water to the cuvette, adjusting zero, adding the reaction solution to the cuvette, and clicking detection to obtain the fluorescence signal value.

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