Zhang Ni, Cheng Huijuan, Liu Yueling
State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, P. R. China.
Department of Applied Biology, East China University of Science and Technology, Shanghai 200237, P. R. China.
Anal Chem. 2024 Sep 16. doi: 10.1021/acs.analchem.4c03040.
Intracellular potassium ions play a crucial role in cellular homeostasis associated with physiological and pathological processes. It is still challenging but definitely crucial to precisely and dynamically image subcellular K. Herein, the first mitochondria-targeted DNA tweezers (KLA-tweezers) were developed for the fluorescence imaging of mitochondrial K with high selectivity and accuracy. The proposed KLA-tweezers consisted of two DNA double-crossover (DX) motifs, a K-aptamer, and a mitochondria-targeted peptide (KLA) which was conjugated at the rear of DX arms via complementary base pairing. Upon contact with K, the K-aptamer experienced a conformational change from an open-chain G-rich sequence to a G-quadruplex with a compact conformation, which was reflected by the Förster resonance energy transfer process between Cy3 and Cy5 labeled at the end of the DX arms. The open and closed states of the tweezers before and after KLA modification were confirmed by gel electrophoresis and fluorescence spectra together with atomic force microscopy (AFM) and transmission electron microscopy (TEM) images, suggesting the KLA assembly had no effect on the morphology change. The proposed tweezers and KLA-tweezers showed low cytotoxicity, excellent selectivity, and good reversibility upon alternating addition of 18-crown-6 and K. Besides, the KLA-tweezers exhibited outstanding stability and accurate mitochondria location ability. Upon stimulation of ATP or nigericin, intracellular fluorescence imaging of mitochondrial K dynamics was successfully achieved. This strategy has broad prospects as a general optical sensing platform for other metal ions or organelles in living cells.
细胞内钾离子在与生理和病理过程相关的细胞稳态中起着至关重要的作用。精确且动态地对亚细胞水平的钾进行成像仍然具有挑战性，但绝对至关重要。在此，首次开发了靶向线粒体的DNA镊子（KLA镊子），用于对线粒体钾进行高选择性和准确性的荧光成像。所提出的KLA镊子由两个DNA双交叉（DX）基序、一个钾适配体和一个靶向线粒体的肽（KLA）组成，该肽通过互补碱基配对连接在DX臂的末端。与钾接触后，钾适配体经历了从开放链富含鸟嘌呤序列到具有紧凑构象的G-四链体的构象变化，这通过在DX臂末端标记的Cy3和Cy5之间的Förster共振能量转移过程得以体现。通过凝胶电泳、荧光光谱以及原子力显微镜（AFM）和透射电子显微镜（TEM）图像共同证实了KLA修饰前后镊子的开放和闭合状态，表明KLA组装对形态变化没有影响。所提出的镊子和KLA镊子在交替添加18-冠-6和钾时表现出低细胞毒性、优异的选择性和良好可逆性。此外，KLA镊子表现出出色的稳定性和准确的线粒体定位能力。在ATP或尼日利亚菌素刺激下，成功实现了线粒体钾动力学的细胞内荧光成像。作为一种用于活细胞中其他金属离子或细胞器的通用光学传感平台，该策略具有广阔的前景。
