A FRET biosensor constructed using pH sensitive G-quadruplex DNA for detecting mitochondrial autophagy

Mitochondria are crucial powerhouses and central organelles for maintaining normal physiological activities in eukaryotic cells. The use of highly specific optical biosensors to monitor mitochondrial autophagy (mitophagy) is an important way for detecting mitochondrial abnormalities. Herein, we report a pH responsive G-quadruplex (G4) structure folded by the oligonucleotide named P24. P24 is composed of four GGCCTG repeating units, and the high guanine content allows it to form an antiparallel G4 topology at pH 4.5 (lysosomal pH). However, when pH increases to around 7.4 (mitochondrial pH), P24 further transforms into a double-stranded structure. Unlike most oligonucleotides that enter lysosomes, P24 highly targets mitochondria in live cells. These characteristics enable P24 to construct a pH responsive optical biosensor by linking a pair of fluorescence resonance energy transfer (FRET) fluorophores. The P24 based biosensor demonstrates reliable applications in detecting mitophagy in live cells.