Zhao Rujian, Xiao Yao, Tang Yidan, Lu Baiyang, Li Bingling
State Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Science, Changchun, Jilin 130022, China.
University of Science and Technology of China, Hefei, Anhui 230026, China.
ACS Sens. 2024 Sep 27;9(9):4803-4810. doi: 10.1021/acssensors.4c01233. Epub 2024 Sep 16.
CRISPR/Cas12a has been widely used in molecular diagnostics due to its excellent trans-cleavage activity. However, conventional reporters, such as F/Q-labeled single-stranded DNA (ssDNA) reporters, enzyme-labeled reporters, and spherical nucleic acid reporters, require complex modification or labeling processes. In this study, we have developed a rapid, universal, and label-free CRISPR/Cas12a-based biomarker detection platform via designing a G-quadruplex (G4) containing a hairpin structure as the reporter. The hairpin loop design of hairpin G4 improves the cleavage efficiency of Cas12a and the signal strength of the G4 binding ligand. Meanwhile, the incorporation of a G4 binding dye (protoporphyrin IX) eliminates the need for complex modifications. The CRISPR-hairpin G4 detection platform is capable of detecting ssDNA, double-stranded DNA, genetic RNAs, and miRNAs. Moreover, this platform achieves label-free detection in clinical samples, demonstrating its practical applicability and efficiency.
由于其出色的反式切割活性，CRISPR/Cas12a已在分子诊断中得到广泛应用。然而，传统的报告分子，如F/Q标记的单链DNA（ssDNA）报告分子、酶标记报告分子和球形核酸报告分子，需要复杂的修饰或标记过程。在本研究中，我们通过设计一种含有发夹结构的G-四链体（G4）作为报告分子，开发了一种快速、通用且无需标记的基于CRISPR/Cas12a的生物标志物检测平台。发夹G4的发夹环设计提高了Cas12a的切割效率和G4结合配体的信号强度。同时，引入G4结合染料（原卟啉IX）消除了复杂修饰的需要。CRISPR-发夹G4检测平台能够检测ssDNA、双链DNA、遗传RNA和miRNA。此外，该平台在临床样本中实现了无标记检测，证明了其实际适用性和效率。
