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CN-121975938-A - CRISPR/Cas12 a-responded DNA hydrogel amplification-free detection method for miRNA and application

CN121975938ACN 121975938 ACN121975938 ACN 121975938ACN-121975938-A

Abstract

The invention belongs to the field of biological detection, and provides a method for detecting miRNA by DNA hydrogel amplification-free in CRISPR/Cas12a response and application thereof. The method mainly comprises the following steps of (1) preparing fluorescence quenched DNA hydrogel by using a DNA1/2 chain modified by a fluorescent group FAM or a quencher BHQ1 respectively and a Linker chain complementary paired with partial bases of the two DNA chains, (2) incubating Cas12a, crRNA and a target miRNA-21 in a buffer solution to obtain an activated CRISPR/Cas12a system complex, and adding the CRISPR/Cas12a complex into the prepared DNA hydrogel to obtain the cut hydrogel which can display fluorescence signals again. The invention does not need to amplify or concentrate the target to be detected, has simple operation, high sensitivity and accurate detection result, and can be used for detecting the tumor marker miRNA.

Inventors

  • WANG ZHAOYIN
  • SUN RUI

Assignees

  • 南京师范大学

Dates

Publication Date
20260505
Application Date
20260204

Claims (10)

  1. 1. A method for detecting miRNA by using CRISPR/Cas12a responding DNA hydrogel amplification-free, which is characterized by comprising the following steps: Step 1, preparing polyacrylamide chains PS1 and PS2: polymerizing DNA1/DNA2 chains modified with acrylamide groups and FAM/BHQ1 into a polyacrylamide chain PS1/PS2 containing a plurality of DNA branched chains under the catalysis of an initiator; step 2, preparing fluorescence quenching DNA hydrogel: Mixing the polyacrylamide chain prepared in the step 1 with a Linker chain, and obtaining fluorescence quenched DNA hydrogel by a base complementary pairing method; step 3, cutting hydrogel: Incubating Cas12a protein, crRNA and matched buffer solution to obtain Cas12a/crRNA complex, then mixing with target miRNA-21 to activate trans-cleavage activity of CRISPR/Cas12a system, adding the obtained mixed solution into DNA hydrogel prepared in step 2 to react to obtain cleaved hydrogel Step 4, fluorescence detection: Fluorescence signal detection was performed on the cut DNA hydrogel using a fluorescence spectrometer.
  2. 2. The method according to claim 1, wherein in step 1, the polyacrylamide strands PS1 and PS2 are prepared by placing a mixed solution of the primer DNA1/DNA2, acrylamide and TAE/Mg 2+ buffer and a newly prepared solution of ammonium persulfate APS and tetramethyl ethylenediamine TEMED in a vacuum oven at 37℃for 15 min, then adding the APS and TEMED solutions to the DNA mixed solution, and then placing 20min in the vacuum oven to obtain PS1/PS2.
  3. 3. The method of claim 2, wherein the nucleotide sequence of the DNA1 strand is 5'-FAM-TCACAGATGAGTATCTTTTATTT-Acrydite-3', and the nucleotide sequence of the DNA2 strand is 5'-Acrydite-TTTATTTTCTTGTCTCCCGAGAT-BHQ1-3'.
  4. 4. The method of claim 1, wherein in step 2, the fluorescence-quenched DNA hydrogel is prepared by mixing PS1, PS2 and Linker solutions in a molar ratio of 1:1:1 with vigorous shaking, placing 5min at 60 ℃, and then cooling the mixture to room temperature to form the DNA hydrogel.
  5. 5. The method of claim 4, wherein the Linker chain nucleotide sequence is 5'-GATACTCATCTGTGATTATTATCTCGGGAGACAAG-3'.
  6. 6. The method of claim 1, wherein in step 3 Cas12a, crRNA and 1 xnebuffer are incubated at 37 ℃ for 10 min to give Cas12a/crRNA complex, then target miRNA21 is added and incubation at 37 ℃ is continued for 5min, then activated CRISPR/Cas12a system mixed solution is added to a centrifuge tube containing DNA hydrogel and incubated 60-80 min with shaking in a 37 ℃ shaker.
  7. 7. The method of claim 6, wherein the crRNA nucleotide sequence is 5'-UAAUUUCUACUAAGUGUAGAUUCAACAUCAGUCUGAUAAGCUA-3', miRNA and the nucleotide sequence is 5'-UAGCUUAUCAGACUGAUGUUGA-3'.
  8. 8. The method of claim 1, wherein in step 4, the fluorescence spectrum of the hydrogel solution is measured using a fluorescence spectrometer in the range of 500-640 nm, and the conditional excitation wavelength is set to 480 nm and the emission wavelength is set to 520 nm.
  9. 9. The method of claim 7, wherein the linear regression equation is derived by plotting the fluorescence intensity of the cut hydrogel solution versus the concentration of miRNA-21.
  10. 10. The use of the method according to any one of claims 1 to 7, wherein a sample of unknown concentration is added to the kit and the detected fluorescence intensity is substituted into the linear regression equation to obtain the miRNA-21 concentration of the sample.

Description

CRISPR/Cas12 a-responded DNA hydrogel amplification-free detection method for miRNA and application Technical Field The invention belongs to the field of biological detection and sensing, and particularly relates to a CRISPR/Cas12a response-based DNA hydrogel amplification-free rapid detection method for miRNA and application thereof. Background Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and adjacent CRISPR-associated proteins (Cas) are derived from the adaptive immune system produced by prokaryotes, and CRISPR/Cas systems play an important role in the field of gene editing due to their powerful ability to locate and cleave nucleic acids. With the continuous and intensive research of scientific researchers, the CRISPR/Cas system also provides a powerful tool for the development of biosensing technology. Among them, cas12a (Cpf 1) is an RNA-mediated DNA nuclease, and Cas12a is attracting attention in the field of nucleic acid detection due to its advantages of wide target range, strong cleavage activity, and the like. At present, the CRISPR/Cas12a system is widely applied to the professional fields of disease diagnosis and treatment, food safety, environment detection and the like, and has good application prospect. However, most of the current research is based on ssDNA/dsDNA to activate trans-cleavage activity of CRISPR/Cas12a, and for RNA targets, the integration of nucleic acid reverse transcription amplification technology with CRISPR/Cas12a system cannot be directly used for RNA detection, which greatly increases complexity of operation, professional requirements and detection cost. Malignant tumors have become a major challenge in the world public health field, severely threatening human life health and quality of life. Early diagnosis, accurate treatment and prognosis evaluation are key to improving survival rate of cancer patients, and efficient detection of tumor markers is a core technical support for achieving the above objective. MicroRNA (miRNA) is taken as an endogenous single-stranded non-coding RNA with the length of about 18-25 nucleotides, and plays a core regulation role in key biological processes such as cell growth, differentiation, apoptosis and the like. Various studies indicate that abnormal expression of miRNAs is closely related to tumorigenesis, development, metastasis and invasion, and has become a very potential tumor marker. miRNA-21 is one of the earliest detected and widely studied miRNAs, is highly expressed in various malignant tumor tissues and body fluids such as lung cancer, breast cancer, brain glioma and the like, is hopeful to realize early noninvasive screening of tumors by detecting the miRNA-21 level in the body fluids, and has extremely high clinical application value. However, the inherent characteristics of the short sequence of miRNA-21, the high similarity of sequences among members of the same group, and extremely low expression level make the development of sensitive, accurate and efficient detection techniques a great challenge. Utilizing the nonspecific cleavage activity of CRISPR/Cas12a, researchers have developed a variety of highly sensitive miRNA-21 detection platforms, such as Wang Jing team combining Rolling Circle Amplification (RCA) with CRISPR/Cas12a systems, developed an ultrasensitive Surface Enhanced Raman Scattering (SERS) platform, with RCA providing target-dependent amplification with strict sequence discrimination through padlock probe ligation, while CRISPR/Cas12a systems promote stable signal generation through trans cleavage activity, representing their great potential in noninvasive early lung cancer diagnosis (article: al chem 2025, 97, 38, 21098-21105). However, the traditional CRISPR/Cas12a system still has a key bottleneck in miRNA detection, namely, the traditional CRISPR/Cas12a system strictly depends on a nucleic acid amplification method and can not directly identify an RNA target, so that the operation complexity and detection error are increased, and the simplification requirement of amplification-free detection is violated. The DNA hydrogel is used as a three-dimensional network structure material formed by taking a DNA chain as a cross-linking agent through base complementation pairing, has good biocompatibility, biodegradability and structural designability, and the gel-sol phase transformation process can be accurately regulated and controlled through nucleic acid specific hybridization or enzyme digestion reaction, so that the DNA hydrogel has been widely applied to the field of biosensing. The advantages of the DNA hydrogel and the CRISPR/Cas12a system are combined, the bottleneck of the existing miRNA detection technology is hopeful to be broken through, the detection flow is simplified, and the direct, rapid and accurate detection of miRNA is realized. Disclosure of Invention Based on the defects and clinical detection requirements of the prior art, the invention aims to provide a CRISPR/Cas12