Wei Xiaoling, Ye Muling, Jia Haiyan, Zhou Xiaoyu, Wang Yujie, Li Mengru, Xue Chang, Xu Liang, Shen Zhifa
Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, Cixi Biomedical Research Institute, School of Laboratory Medicine and Life sciences, Wenzhou Medical University, Wenzhou, 325035, China.
Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, Cixi Biomedical Research Institute, School of Laboratory Medicine and Life sciences, Wenzhou Medical University, Wenzhou, 325035, China.
Biosens Bioelectron. 2025 Mar 1;271:116975. doi: 10.1016/j.bios.2024.116975. Epub 2024 Nov 26.
Aberrant microRNA (miRNA) expression is frequently implicated in various cancers, making the monitoring of intracellular miRNA levels a promising strategy for cancer diagnosis and therapy. However, detecting miRNA with high precision and sensitivity at the cellular level remains challenging due to its small size and low abundance. In this study, we attached hydrophobic cholesterol molecules to hydrophilic DNA chains to self-assemble into cholesterol-DNA micelles. The products of rolling ring amplification were linked to the surface of cholesterol-DNA, and two hairpins (H1 and H2) used for hybridization chain reaction (HCR) were simultaneously tethered to the branch, ultimately forming the assembled nanoprobe (RC-HCR) with signal amplification for detecting and imaging miRNA in living cells. This design significantly increased the concentration of HCR hairpins and also shortened their physical distance, thereby enhancing kinetics and signal amplification. Moreover, we demonstrated that the lipid particles could be assembled by simply stirring in a buffered solution, allowing the system to enter cells naturally. Using miR-21 as the model target, we found that the RC-HCR probe had a detection limit of 1 fM and a wide quantitative range (1 fM to 80 nM) at 37 °C within 0.5 h. In addition, RC-HCR exhibited high selectivity for miRNA detection and could accurately identify wild-type miR-21 from its mutants and other miRNAs. Furthermore, we showed that RC-HCR could efficiently image miR-21 in living cells. Collectively, our strategy provides a valuable nanoprobe for detecting and imaging miRNAs in live cells, highlighting a novel tool for early clinical diagnosis.
异常的微小RNA（miRNA）表达常常与各种癌症相关，这使得监测细胞内miRNA水平成为癌症诊断和治疗的一种有前景的策略。然而，由于其尺寸小和丰度低，在细胞水平上高精度和高灵敏度地检测miRNA仍然具有挑战性。在本研究中，我们将疏水性胆固醇分子连接到亲水性DNA链上，使其自组装成胆固醇-DNA胶束。滚环扩增产物连接到胆固醇-DNA表面，用于杂交链式反应（HCR）的两个发夹（H1和H2）同时连接到分支上，最终形成具有信号放大功能的组装纳米探针（RC-HCR），用于检测和成像活细胞中的miRNA。这种设计显著提高了HCR发夹的浓度，还缩短了它们的物理距离，从而增强了动力学和信号放大。此外，我们证明了脂质颗粒可以通过在缓冲溶液中简单搅拌来组装，使该系统能够自然进入细胞。以miR-21作为模型靶点，我们发现在37℃下0.5小时内，RC-HCR探针的检测限为1 fM，定量范围宽（1 fM至80 nM）。此外，RC-HCR对miRNA检测具有高选择性，能够从其突变体和其他miRNA中准确识别野生型miR-21。此外，我们表明RC-HCR可以在活细胞中高效地对miR-21进行成像。总的来说，我们的策略为检测和成像活细胞中的miRNA提供了一种有价值的纳米探针，突出了一种用于早期临床诊断的新型工具。
