Protease-Triggered, Spatially Controlled DNA Assembly in Apoptotic Cells for Early Evaluation of Therapeutic Efficacy

Despite numerous advances in the use of DNA as building blocks to assemble complex structures, the dearth of strategies that allow for protease-controlled in situ DNA assembly in living cells remains a bottleneck in this field. Here, we present a modular engineering approach to achieve protease-triggered self-assembly of DNA in apoptotic cells for early evaluation of tumor response to drug treatment. In the design, peptide nucleic acid is introduced as a building bridge to engineer DNA building blocks with peptides and thus to suppress their self-assembly activity, while caspase-3 (Casp-3) protease-mediated enzymatic cleavage of the peptide substrate enables the activation of the DNA assembly, generating fluorescence signal output for real-time monitoring of Casp-3 activity. Furthermore, the specific protease triggering imparts DNA assembly with spatial selectivity to apoptotic cells in vivo, allowing for early evaluation of tumor therapeutic efficacy. Moreover, the strategy is extended to probe the activity of MMP-2 for lymph node metastasis imaging, demonstrating the universality of this approach. This work highlights protease-controlled DNA assembly in ways that are simple and versatile, with the potential to expand the repertoire of DNA nanotechnology for diverse biomedical applications.