Proteomic Exploration of Endocytosis of Framework Nucleic Acids

Efficient cell internalization of framework nucleic acid nanostructures free of transfection agents provides new opportunities for developing biocompatible and intelligent nanoprobes and drug delivery carriers. Here, a proteomic identification method to screen target proteins that interact with tetrahedral DNA nanostructures (TDNs) during the process of endocytosis by combining drug affinity responsive target stability (DARTS) with liquid chromatography/tandem mass spectrometry (LC-MS/MS) techniques, is reported. It is found that that caveolin-1 (CAV1) and macropinocytosis-related protein sorting nexin5 (SNX5) are associated with the endocytosis of TNDs, which is further validated by microscale thermophoresis (MST) analysis. CAV1- and SNX5- knockout experiments reveal that both caveolae-mediated endocytosis and macropinocytosis mediate the cellular uptake of TDNs, which complement previous findings with fluorescence tracing methods. This method provides a generic strategy to analyze cellular internalization process of DNA nanostructures for biomedical applications.