Cell‐Type‐Specific Prediction of DNA Duplex Stability Guides Functional DNA Operation Along Cancer Progression

ABSTRACT Functional DNAs have attracted attention in cancer diagnosis and therapy owing to their programmable sequence specificity. However, the designed DNAs often fail to operate in target cells because intracellular environments evolve during cancer progression and across different cell types. Here, we aimed to predict DNA duplex stability across stages of cancer progression using thermodynamic parameters. As direct physicochemical measurements in living cells are challenging, we employed a pseudo‐cellular System for Highlighting the Environments inside the ceLL (SHELL) that preserves native cellular components and molecular crowding. We determined the thermodynamic parameters of DNA duplex formation in SHELLs derived from normal, mild cancer, and aggressive cancer cells. DNA duplexes became progressively destabilized with cancer progression, leading us to derive exact cell‐type‐specific nearest‐neighbor (NN) parameters in SHELL (SHELL‐NN parameters). As an example of how these parameters can be applied, we identify a quantitative design criterion in which duplex regions must be sufficiently stabilized, defined by duplex stability at 37°C (−∆ G ° 37 > 9.8 kcal mol −1 ) to remain hybridized in normal cells and suppress background dissociation, while still enabling target‐induced dissociation in cancer cells. This environment‐specific thermodynamic threshold provides a quantitative design criterion for functional nucleic acid systems in cancer‐associated cellular environments.