DNA-Based pH Nanosensor with Adjustable FRET Responses to Track Lysosomes and pH Fluctuations

Extensive attention has been recently focused on designing signal adjustable biosensors. However, there are limited approaches available in this field. In this work, to visually track lysosomes with high contrast, we used the i-motif structure as a pH-responsive unit and proposed a novel strategy to regulate the fluorescence resonance energy transfer (FRET) response of the pH sensor. By simply splitting the i-motif into two parts and modulating the split parameters, we can tune the pH transition midpoint (pHt) from 5.71 to 6.81 and the signal-to-noise ratio (S/N) from 1.94 to 18.11. To facilitate the lysosome tracking, we combined the i-motif split design with tetrahedral DNA (Td). The obtained pH nanosensor (pH-Td) displays appropriate pHt (6.12) to trace lysosomes with high S/N (10.3). Benefited from the improved stability, the superior cell uptake and lysosomal location of pH-Td, the visualization of the distribution of lysosomes, the lysosome–mitochondria interaction, and the pH changes of lysosomes in response to different stimuli were successfully achieved in NIH 3T3 cells. We believe that the design concept of controlling the split sequence distance will provide a novel insight into the design of i-motif-based nanosensors and even inspire the construction of smart DNA nanodevices for sensing, disease diagnosis, and controllable drug delivery.