Recently, Professor Cheng Mingpan's research group at our university published a research paper titled “i-Motif DNA Molecular Beacon for MicroRNA Detection” in Nucleic Acids Research, an internationally renowned journal in the field of nucleic acid research. Luo Tengshuo, a doctoral student in the School of Engineering in the class of 2022, is the first author of the paper, Professor Cheng Mingpan is the corresponding author, and China Pharmaceutical University is the sole corresponding institution.
Nucleic acid molecular beacons (Molecular Beacons) are an important class of nucleic acid detection probes due to their simple structure, low background signal, high sensitivity, and specificity. They feature a hairpin-like stem-loop structure, where the stem is typically composed of double-stranded DNA with reverse complementary pairing, and the single-stranded loop region serves as the primary target recognition unit. When the molecular beacon binds to the target molecule, the increase in molecular rigidity causes the double-stranded stem structure to unwind, thereby generating a signal.
The Cheng Mingpan research group has extensive experience in studying the structural diversity and stability of i-motif DNA (JACS, 2021; Angew, 2021). An i-motif is a four-stranded nucleic acid structure formed by the folding of cytosine-rich sequences. The foundation of the i-motif structure is the stacking of semi-protonated cytosine-cytosine base pairs. Unlike double-stranded structures, the stability of i-motif structures is closely related to environmental pH. i-Motif DNA is widely present at critical chromosomal locations, such as telomere ends and promoters of cancer-related genes, and plays an important role in maintaining chromosomal stability and regulating gene expression. On the other hand, as an environmentally sensitive nucleic acid, it can serve as a basic structural unit for dynamic DNA-based nanotechnology, with applications in controlled drug release and biosensing.
Recently, the research group reported the first molecular beacon (iMB) based on the i-motif DNA structure and successfully applied it to the detection of cancer biomarkers microRNA (miRNA). In the iMB structure designed in this study, the i-motif serves as the stem region of the molecular beacon, functioning both as the structural framework and a pH sensor, while the three single-stranded loop regions act as recognition units directly involved in binding to the target miRNA. To address the issue of simultaneous environmental pH and temperature effects on iMB structural stability, the study utilized nucleic acid phase diagram analysis to optimize and elucidate the dynamic binding equilibrium between target miRNA and iMB, achieving highly sensitive and specific nucleic acid detection. Additionally, the study found that iMB exhibits resistance to nucleases, and this property was leveraged to design an isothermal signal amplification technique, further enhancing detection sensitivity. Finally, iMB was applied to miRNA detection in model cells and cancer blood samples, with results showing performance comparable to that of classical RT-qPCR detection.
This study was guided and supported by Jean-Louis Mergny from École Polytechnique, Professor Zhou Jun from Nanjing University, and Associate Professor Liu Yunlong from the School of Engineering at China Pharmaceutical University. This work was supported by the National Natural Science Foundation of China, the Jiangsu Provincial Natural Science Foundation, the Jiangsu Distinguished Professor Program, the Jiangsu Provincial Frontier Technology Research and Development Program, the Jiangsu Provincial Innovation and Entrepreneurship Team Program, and our university's High-Level Talent Program.
Full text link: https://doi.org/10.1093/nar/gkaf556.
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