Programmable DNA Nanostructures for hTERT Compartmentalization and Translocation in Living Cells

Abstract Protein translocation is essential for cellular function, and compartmentalizing proteins can regulate their activity. However, constructing artificial intracellular compartments to control this process remains a significant challenge. Herein, we report the artificial dynamic assembly of DNA condensates in the cytoplasm, enabling the specific compartmentalization of human telomerase reverse transcriptase (hTERT) and effectively inhibiting its canonical and non‐canonical activities. DNA‐based condensates are formed through the dynamic assembly of branched DNA structures incorporating a mitochondria‐targeting triphenylphosphine and a telomerase primer for telomerase recognition. Upon uptake by cancer cells, the primer interacts with telomerase, triggering a strand displacement reaction that releases X‐shaped DNA. The sticky palindromic sequences in the X‐shaped DNA promote self‐assembly, forming DNA condensates on mitochondria. These condensates disrupt mitochondria functions, increasing reactive oxygen species (ROS) and stimulating the export of hTERT from the nucleus to the cytoplasm. Once in the cytoplasm, hTERT is specifically captured by the DNA condensates, preventing its translocation to mitochondria. The reduction of hTERT in both nucleus and mitochondria further results in impaired cellular proliferation and mitochondrial dysfunction in cancer cells. This work provides a highly controllable strategy for manipulating protein translocation through compartmentalization in living cells, offering a promising new avenue for modulating cellular behavior.