Wang Ya-Xin, Wang Dong-Xia, Ma Jia-Yi, Wang Jing, Du Yi-Chen, Kong De-Ming
State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, College of Chemistry, Nankai University, Tianjin, 300071, PR China.
Analyst. 2021 Apr 26;146(8):2600-2608. doi: 10.1039/d1an00275a.
Accurate and specific analysis of adenosine triphosphate (ATP) expression levels in living cells can provide valuable information for understanding cell metabolism, physiological activities and pathologic mechanisms. Herein, DNA nanolantern-based split aptamer nanoprobes are prepared and demonstrated to work well for in situ analysis of ATP expression in living cells. The nanoprobes, which carry multiple split aptamer units on the surface, are easily and inexpensively prepared by a "one-pot" assembly reaction of four short oligonucleotide strands. A series of characterization experiments verify that the nanoprobes have good monodispersity, strong biostability, high cell internalization efficiency, and fluorescence resonance energy transfer (FRET)-based ratiometric response to ATP in the concentration range covering the entire intracellular ATP expression level. By changing the intracellular ATP level via different treatments, the nanoprobes are demonstrated to show excellent performance in intracellular ATP expression analysis, giving a highly ATP concentration-dependent ratiometric fluorescence signal output. ATP-induced formation of large-sized DNA aggregates not only amplifies the FRET signal output, but also makes in situ ATP-imaging analysis in living cells possible. In situ responsive crosslinking of nanoprobes also makes them capable of lighting up the mitochondria of living cells. By simply changing the split aptamer sequence, the proposed DNA nanolantern-based split aptamer strategy might be easily extended to other targets.
准确且特异性地分析活细胞中三磷酸腺苷（ATP）的表达水平可为理解细胞代谢、生理活动及病理机制提供有价值的信息。在此，制备了基于DNA纳米灯笼的分裂适体纳米探针，并证明其在活细胞中ATP表达的原位分析中表现良好。这些纳米探针在表面携带多个分裂适体单元，通过四条短寡核苷酸链的“一锅法”组装反应即可轻松且低成本地制备。一系列表征实验证实，纳米探针具有良好的单分散性、强生物稳定性、高细胞内化效率，以及在覆盖整个细胞内ATP表达水平的浓度范围内基于荧光共振能量转移（FRET）的对ATP的比率响应。通过不同处理改变细胞内ATP水平，纳米探针在细胞内ATP表达分析中表现出优异性能，给出高度依赖ATP浓度的比率荧光信号输出。ATP诱导形成的大尺寸DNA聚集体不仅放大了FRET信号输出，还使活细胞中的原位ATP成像分析成为可能。纳米探针的原位响应交联还使其能够照亮活细胞的线粒体。通过简单改变分裂适体序列，所提出的基于DNA纳米灯笼的分裂适体策略可能很容易扩展到其他靶标。
