Method for detecting miRNA through ratio-type CRISPR / Cas12a system

Abstract

The invention relates to a method for detecting miRNA (micro Ribonucleic Acid) by using a ratio-type CRISPR / Cas12a (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR / Cas12a) system. The method is characterized by being realized by applying a multifunctional probe in the CRISPR / Cas12a system. Wherein the multifunctional probe is a single-stranded DNA (deoxyribonucleic acid) with a hairpin structure and comprises two functional areas, namely a neck part and a loop part; wherein the neck part of the probe is used for identifying a target miRNA; wherein the probe ring part has various different functions of lightening a fluorescent dye, activating a CRISPR / Cas12a system, serving as a Cas12a trans-cracking substrate, triggering a self-driven cascade reaction and the like; the working principle of the method is as follows: when no target exists, the ring part of the probe cannot activate the CRISPR / Cas12a system, the G quadruplex structure of the ring part of the probe can be combined with a fluorescent dye to emit fluorescence, and the neck part of the probe is opened by combining the sticky end with the target when the target exists, so that the G quadruplex structure of the ring part of the probe disappears and releases an activator to activate the CRISPR / Cas12a system; the activated Cas12a simultaneously reversely cuts the reporter probe and the multifunctional probe around the Cas12a, so that the fluorescence of the reporter probe is enhanced and the fluorescence of the multifunctional probe is reduced, and the multifunctional probe releases more activators to trigger a self-driven cascade reaction, so that the fluorescence of the reporter probe is further enhanced and the fluorescence of the multifunctional probe is further reduced; quantitative detection of the target miRNA can be realized through the mathematical relationship between the fluorescence ratio of the report probe and the multifunctional probe and the target concentration. The ratio-type CRISPR / Cas12a system is designed according to the structure activity of DNA, the property of the fluorescent dye and the characteristics of the CRISPR / Cas12a system, sensitive detection of miRNA is achieved, and the ratio-type CRISPR / Cas12a system can be used for scientific research analysis and clinical detection and has wide market prospects.

Description

Technical Field

[0001] The present invention relates to an analysis method, specifically to a method for detecting miRNA by a ratio-type CRISPR / Cas12a system. Background Art

[0002] MicroRNA (miRNA) is a class of endogenous non-coding RNAs with a length of about 22 nucleotides. By specifically binding to the 3'-untranslated region (3'-UTR) of target mRNA, it regulates gene expression at the post-transcriptional level and plays a key role in important life activities such as cell cycle regulation, cell differentiation, and apoptosis. Recent studies have shown that abnormal miRNA expression patterns are closely related to the occurrence and development of various diseases, including malignant tumors, cardiovascular diseases, and neurodegenerative diseases such as Alzheimer's disease. Its unique tissue expression specificity and stability in peripheral blood make it a highly potential new molecular diagnostic marker with broad development prospects. Constructing a highly sensitive and specific miRNA detection technology system has important clinical significance for the early diagnosis of diseases, monitoring of treatment effects, and realization of precision medicine.

[0003] Currently, the technical platform based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) is still the commonly used standard method for miRNA detection. This technology converts miRNA into complementary DNA (cDNA) through reverse transcription reaction, and then performs real-time fluorescence quantitative analysis. However, this technology has some limitations. On the one hand, the reverse transcription efficiency is greatly affected by the secondary structure and GC content of miRNA, which is likely to cause deviation in quantitative results; on the other hand, the qPCR process requires precise temperature control equipment equipped in professional laboratories, resulting in a high degree of professional dependence and detection cost. In addition, the primer design for different miRNAs needs to be optimized repeatedly, which is difficult to meet the requirements of high-throughput detection. These technical defects have greatly limited the wide application of this technology in primary medical institutions and point-of-care testing (POCT) scenarios.

[0004] The CRISPR / Cas12a system provides new opportunities for the innovation of molecular diagnostic techniques with its unique dual nuclease activities (specific recognition and trans-cleavage) and room-temperature reaction characteristics. Its core mechanism is the recognition of target DNA mediated by guide RNA (crRNA), which activates the non-specific single-stranded DNA cleavage activity of the Cas12a protein, and then hydrolyzes the fluorescently labeled reporter probe. However, there are still technical bottlenecks in the existing miRNA detection schemes based on the CRISPR / Cas12a system. On the one hand, it is still necessary to convert miRNA into cDNA and amplify it through RT-PCR. This process not only introduces reverse transcription bias and amplification bias errors, but also increases the operation complexity and detection time. On the other hand, the traditional CRISPR / Cas12a system uses a single fluorescence strategy and is vulnerable to environmental interference, thus reducing the detection accuracy. Ratio-based fluorescence quantitative analysis can eliminate the influence of the environment through a self-calibration or self-reference mechanism and achieve ultrasensitive detection of miRNA.

[0005] To address the above technical bottlenecks, the present invention designs a ratio-based CRISPR / Cas12a system according to the structure-activity relationship of DNA, the properties of fluorescent dyes, and the characteristics of the CRISPR / Cas12a system to achieve sensitive detection of miRNA. Summary of the Invention

[0006] The object of the present invention is precisely to construct a method for detecting miRNA using a ratio-based CRISPR / Cas12a system.

[0007] The object of the present invention can be achieved by the following technical measures:

[0008] A method for detecting miRNA using a ratio-type CRISPR / Cas12a system, characterized in that: the method is realized by applying a multifunctional probe in the CRISPR / Cas12a system; wherein the multifunctional probe is a single-stranded DNA with a hairpin structure, comprising two functional regions, a neck and a loop; wherein the neck of the probe is used for the recognition of target miRNA; wherein the loop of the probe has multiple different functions such as lighting up a fluorescent dye, activating the CRISPR / Cas12a system, serving as a Cas12a trans-cleavage substrate, triggering a self-driven cascade reaction, etc.; the working principle of the method is that in the absence of a target, the loop of the probe cannot activate the CRISPR / Cas12a system, and its G-quadruplex structure can bind to the fluorescent dye and emit fluorescence. When the target is present, the neck of the probe binds to the target through sticky ends and opens, resulting in the disappearance of the G-quadruplex structure of the probe loop, releasing an activator to activate the CRISPR / Cas12a system. The activated Cas12a will simultaneously trans-cleave the surrounding reporter probe and the multifunctional probe, resulting in an increase in the fluorescence of the reporter probe and a decrease in the fluorescence of the multifunctional probe. At the same time, the multifunctional probe releases more activators to trigger a self-driven cascade reaction, further enhancing the fluorescence of the reporter probe and further decreasing the fluorescence of the multifunctional probe. The quantitative detection of target miRNA can be achieved through the mathematical relationship between the fluorescence ratio of the reporter probe and the multifunctional probe and the target concentration.

[0009] The present invention has the following beneficial effects:

[0010] The present invention constructs a direct detection system for miRNA based on the CRISPR / Cas12a system, breaking through the inherent technical path of traditional methods that rely on reverse transcription to generate cDNA, effectively eliminating the reverse transcription pretreatment step and the accompanying sequence preference interference, simplifying the detection steps while reducing the detection cost.

[0011] The present invention constructs a ratio-type CRISPR / Cas12a system by using a multifunctional probe, avoiding the problem that the fluorescence signal in traditional single-fluorescence strategies is easily affected by the environment and improving the detection accuracy.

[0012] The multifunctional probe used in the present invention has multiple different functions such as sensing the target, lighting up the fluorescent dye, activating the CRISPR / Cas12a system, serving as a Cas12a trans-cleavage substrate, triggering a self-driven cascade signal amplification, etc. Through the design of integrating target sensing and signal amplification, it not only reduces the background signal and enhances the detection signal, but also saves the detection time and reduces the detection cost. It can be widely applied to scientific research and clinical laboratories and has broad market prospects. Specific embodiments

[0013] The present invention will be further described below in conjunction with embodiments, but does not limit the present invention.

[0014] Example 1

[0015] In this example, the ratio-type CRISPR / Cas12a system method was used to detect miRNA-21, which consisted of five steps: nucleic acid sequence design and synthesis, target recognition, CRISPR / Cas12a reaction, inactivation, and fluorescence detection. The specific steps are as follows:

[0016] (1) Nucleic acid sequence design and synthesis: According to the sequence characteristics of miRNA-21 and the auramine O fluorescent dye aptamer and the characteristics of the present invention, the following sequences need to be designed and synthesized:

[0017] Multifunctional probe: 5’-TCAACATCAGTCTGATAAGCTACGGGGGAGGGTGTGTGGTCTTGCTTGGTTCGTAGCTTATCAGACT-3’

[0018] crRNA: 5’-UAAUUUCUACUAAGUGUAGAUAACCAAGCAAGACCACACAC-3’

[0019] Report probe: BHQ-TTATT-Cy5;

[0020] (2) Target recognition: Take 1 μL of the sample containing miR-21 and 1 μL of the multifunctional probe with a concentration of 20 μM and mix them in a 0.2 mL centrifuge tube containing 8 μL of buffer (20 mM Tris-HCl, 20 mM KCl, 25 mM MgCl 2 , pH 7.4)), incubate at 25 °C for 30 minutes, allow miR-21 to bind to the multifunctional probe and open the hairpin structure, and expose the activator in the probe loop;

[0021] (3) CRISPR / Cas12a reaction: Add 0.5 μL of Cas12a with a concentration of 1 μM, 0.5 μL of crRNA with a concentration of 1 μM, 1 μL of the report probe with a concentration of 10 μM, 2 μL of 10×r2.1 buffer, and 5 μL of the auramine O solution with a concentration of 100 μM, incubate at 25 °C for 30 minutes, allow the exposed activator to activate the CRISPR / Cas12a system, thereby trans-cleaving the multifunctional probe and releasing more activators to form a self-circulating cascade reaction;

[0022] (4) Inactivation: Heat the reaction mixture at 95 °C for 20 minutes and then cool it to 25 °C to inactivate the Cas12a protein to improve the reproducibility of the detection;

[0023] (5) Fluorescence detection: Detect the fluorescence intensity under the conditions of an excitation wavelength of 480 nm and an emission wavelength of 540 nm, and detect the fluorescence intensity under the conditions of an excitation wavelength of 649 nm and an emission wavelength of 670 nm. According to the mathematical relationship between the ratio of the two fluorescence intensities and the concentration of miRNA-21, quantitative detection of miRNA-21 is achieved.

Claims

1. A method for detecting miRNA using a ratiometric CRISPR / Cas12a system, characterized in that: The method is achieved by applying a multifunctional probe in the CRISPR / Cas12a system; wherein the multifunctional probe is a single-stranded DNA probe in a hairpin structure, comprising two functional regions, a neck and a loop; wherein the neck of the probe is a double-stranded DNA structure with a length of 9-30 bp and a sticky end formed by base complementation between the 5' end and the 3' end of the probe, which is used for the recognition of target miRNA; wherein the loop of the probe is a G-quadruplex structure with a length of 30-60 nt, which has multiple different functions such as lighting up fluorescent dyes, activating the CRISPR / Cas12a system, serving as a Cas12a trans-cleavage substrate, triggering a self-driven cascade reaction, etc.; the working principle of the method is that when there is no target, the loop of the probe cannot activate CRISPR The R / Cas12a system has a G quadruplex structure that can bind to fluorescent dyes to emit fluorescence, and the probe neck opens by binding to the target through the sticky end when the target is present, causing the G quadruplex structure of the probe ring to disappear, releasing the activator and activating the CRISPR / Cas12a system. The activated Cas12a will simultaneously trans-cut the reporter probe and the multifunctional probe around it, causing the fluorescence of the reporter probe to increase and the fluorescence of the multifunctional probe to decrease. At the same time, the multifunctional probe releases more activators to trigger a self-driven cascade reaction, further enhancing the fluorescence of the reporter probe and further decreasing the fluorescence of the multifunctional probe. Sensitive detection of target miRNA can be achieved through the mathematical relationship between the fluorescence ratio of the reporter probe and the multifunctional probe and the target concentration.