Decoy DNA Protects Molecular Tension Probes from DNase Degradation

Abstract DNA‐based molecular probes are essential tools for visualizing and quantifying mechanotransduction at the single‐molecule level. However, their application in live‐cell environments is severely limited by DNase‐mediated degradation, which shortens probe lifespan and introduces false‐positive signals. Here, we present a decoy DNA strategy where an excess of unmodified double‐stranded DNA competitively binds DNases, effectively preserving functional DNA probes. This approach extends probe stability from 1–2 h to beyond 24 h, substantially improving signal integrity in live‐cell tension imaging. In contrast to structurally modified nucleic acids, decoy DNA can be readily applied to existing DNA probe systems, enabling seamless integration without the need for additional validation or calibration. This cost‐effective and scalable strategy provides a generalizable framework for stabilizing DNA‐based molecular tools in DNase‐rich environments, enabling high‐precision mechanobiology studies across diverse cell types and extended experiment durations.