Mitochondria-Targeted\nRatiometric KLA-Tweezers for\nFluorescence Imaging of Potassium Ions

Intracellular potassium ions play a crucial role in cellular\nhomeostasis\nassociated with physiological and pathological processes. It is still\nchallenging but definitely crucial to precisely and dynamically image\nsubcellular K<sup>+</sup>. Herein, the first mitochondria-targeted\nDNA tweezers (KLA-tweezers) were developed for the fluorescence imaging\nof mitochondrial K<sup>+</sup> with high selectivity and accuracy.\nThe proposed KLA-tweezers consisted of two DNA double-crossover (DX)\nmotifs, a K<sup>+</sup>-aptamer, and a mitochondria-targeted peptide\n(KLA) which was conjugated at the rear of DX arms via complementary\nbase pairing. Upon contact with K<sup>+</sup>, the K<sup>+</sup>-aptamer\nexperienced a conformational change from an open-chain G-rich sequence\nto a G-quadruplex with a compact conformation, which was reflected\nby the Förster resonance energy transfer process between Cy3\nand Cy5 labeled at the end of the DX arms. The open and closed states\nof the tweezers before and after KLA modification were confirmed by\ngel electrophoresis and fluorescence spectra together with atomic\nforce microscopy (AFM) and transmission electron microscopy (TEM)\nimages, suggesting the KLA assembly had no effect on the morphology\nchange. The proposed tweezers and KLA-tweezers showed low cytotoxicity,\nexcellent selectivity, and good reversibility upon alternating addition\nof 18-crown-6 and K<sup>+</sup>. Besides, the KLA-tweezers exhibited\noutstanding stability and accurate mitochondria location ability.\nUpon stimulation of ATP or nigericin, intracellular fluorescence imaging\nof mitochondrial K<sup>+</sup> dynamics was successfully achieved.\nThis strategy has broad prospects as a general optical sensing platform\nfor other metal ions or organelles in living cells.