State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China; Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, 214063, China.
State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China.
Anal Chim Acta. 2019 Nov 4;1079:146-152. doi: 10.1016/j.aca.2019.06.016. Epub 2019 Jun 10.
A novel versatile locked nucleic acid modified molecular beacon probe (LNA-MB) was developed for imaging intracellular precursor miRNAs (pre-miRNAs) and disturbing Dicer-mediated cleavage process. The target recognition reaction between the smart probe and pre-miRNA can not only induce the conformational changes of probe and block the Dicer cleavage site, but also inhibit the cleavage process, and then achieve down-regulation of miRNA expression. Simultaneously, the target recognition reaction broke the fluorescence resonance energy transfer (FRET) between fluorophore donor FAM and acceptor TAMRA, which were labelled on the LNA-MB probe, further induced the relevant change of fluorescence signal, and then achieved imaging analysis of pre-miRNA and inhibition events in situ. Both in vitro and in single living cell studies showed that the versatile probes exhibited a remarkable performance in targeting with pre-miRNA-21, and nearly 65% downregulation of mature miRNA-21 was achieved with 100 nM probes. All investigations demonstrate that the proposed strategy represents a promising alternative for regulating and inhibiting endogenous disease-associated RNAs, then further for achieving therapeutic outcomes in personalized treatments.
一种新型多功能锁核酸修饰的分子信标探针（LNA-MB）被开发出来，用于对细胞内前体 miRNA（pre-miRNA）进行成像和干扰 Dicer 介导的切割过程。智能探针与 pre-miRNA 之间的靶标识别反应不仅能诱导探针构象变化并阻断 Dicer 切割位点，还能抑制切割过程，从而下调 miRNA 的表达。同时，靶标识别反应破坏了标记在 LNA-MB 探针上的荧光供体 FAM 和荧光受体 TAMRA 之间的荧光共振能量转移（FRET），进一步诱导荧光信号的相关变化，从而实现 pre-miRNA 和原位抑制事件的成像分析。体外和单个活细胞研究均表明，多功能探针在靶向 pre-miRNA-21 方面表现出显著的性能，并且使用 100 nM 探针可实现成熟 miRNA-21 的近 65%下调。所有研究表明，所提出的策略代表了一种有前途的替代方法，可用于调节和抑制与疾病相关的内源性 RNA，从而进一步实现个性化治疗中的治疗效果。
