Mitochondria-Targeted Ratiometric KLA-Tweezers for Fluorescence Imaging of Potassium Ions

• Published in: Analytical chemistry (Washington) 2024-09, Vol.96 (38), p.15355-15363
• Main Authors: Zhang, Ni, Cheng, Huijuan, Liu, Yueling
• Format: Article
• Language: English
• Subjects: Aptamers; Atomic force microscopy; Conformation; Cytotoxicity; Deoxyribonucleic acid; DNA; Electrophoresis; Energy transfer; Fluorescence; Fluorescence resonance energy transfer; Homeostasis; Imaging; Intracellular; Metal ions; Microscopy; Mitochondria; Mitochondrial DNA; Molecular conformation; Nigericin; Organelles; Potassium; Transmission electron microscopy
• ISSN: 0003-2700; 1520-6882; 1520-6882
• 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.