Xu Huo, Zhang Rongbo, Li Feng, Zhou Yingying, Peng Ting, Wang Xuedong, Shen Zhifa
Key laboratory of watershed science and health of Zhejiang Province, Institute of Functional Nucleic Acids and Personalized Cancer Theranostics, Key Laboratory of Laboratory Medicine, Ministry of Education of China, and Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, China.
Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou, 350002, China.
Anal Bioanal Chem. 2016 Sep;408(22):6181-8. doi: 10.1007/s00216-016-9729-z. Epub 2016 Jul 15.
A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related KRAS gene detection based on the one-to-two stoichiometry. During target DNA detection, DHMB can execute signal transduction even if no any exogenous element is involved. Unlike the conventional molecular beacon based on the one-to-one interaction, one target DNA not only hybridizes with one DHMB and opens its hairpin but also promotes the interaction between two DHMBs, causing the separation of two fluorophores from quenchers. This leads to an enhanced fluorescence signal. As a result, the target KRAS gene is able to be detected within a wide dynamic range from 0.05 to 200 nM with the detection limit of 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. Moreover, the point mutations existing in target DNAs can be easily screened. The potential application for target species in real samples was indicated by the analysis of PCR amplicons of DNAs from the DNA extracted from SW620 cell. Besides becoming a promising candidate probe for molecular biology research and clinical diagnosis of genetic diseases, the DHMB is expected to provide a significant insight into the design of DNA probe-based homogenous sensing systems. Graphical Abstract A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related gene KRAS detection based on the one-to-two stoichiometry. Without the help of any exogenous probe, the point mutation is easily screened, and the target DNA can be quantified down to 50 pM, indicating a dramatic improvement compared with traditional molecular beacons.
基于1:2化学计量比开发了一种用于癌症相关KRAS基因检测的强大双发夹分子信标（DHMB）。在靶DNA检测过程中，即使不涉及任何外源元件，DHMB也能执行信号转导。与基于一对一相互作用的传统分子信标不同，一个靶DNA不仅与一个DHMB杂交并打开其发夹结构，还促进两个DHMB之间的相互作用，导致两个荧光团与猝灭剂分离。这导致荧光信号增强。结果，能够在0.05至200 nM的宽动态范围内检测靶KRAS基因，检测限为50 pM，与传统分子信标相比有显著改善。此外，靶DNA中存在的点突变可以很容易地筛选出来。通过对从SW620细胞提取的DNA的PCR扩增产物的分析，表明了其在实际样品中对靶物种的潜在应用。除了成为分子生物学研究和遗传性疾病临床诊断中有前景的候选探针外，DHMB有望为基于DNA探针的均相传感系统的设计提供重要见解。图形摘要基于1:2化学计量比开发了一种用于癌症相关基因KRAS检测的强大双发夹分子信标（DHMB）。无需任何外源探针的帮助，即可轻松筛选点突变，并且靶DNA可以定量至50 pM，与传统分子信标相比有显著改善。
