Intramolecular Orthogonal Reporters for Dissecting Oxidative Stress and Response

Abstract

Molecular response under oxidative stress is a well-known reflection of the development of various diseases. Fluorescent probes for reactive oxygen species (ROS) and for their related molecules integrated into single nanostructures could simultaneously map the localization and abundance of multiple targets with spatiotemporal accuracy in live cells. However, it is extremely challenging because proximity-induced fluorophore interaction leads to low sensitivity and poor specificity. Herein, we engineered intramolecular Orthogonal Reporters (iOR) based on DNA nanostructure scaffolds. Fluorescence reporters in iOR were precisely located with appropriate distance achieved by MD simulation. iOR functionalized with nuclear localization signal peptide is capable of imaging ROS and its related gene repair enzyme (APE1) in nucleus and cytoplasm with spatiotemporal accuracy. iOR discloses the strong positive relation of ROS and APE1 as well as their synergistic regulation. In vivo fluorescence imaging with iOR enables direct observation of ROS and APE1 of the tumor sites of mice. iOR represents a new concept of molecule probes for simultaneous detection of multiple related targets in living cells. We anticipate this simulation-guided probe to find broad applications in cellular biology and nanotechnology.