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The immobilization of enzymes in metal-organic framework (MOF) cages is important in biotechnology. In this context, the mechanism of translocation of proteins through the cavities of the MOF and the roles played by confinement and MOF chemistry in giving rise to stable protein intermediates that are otherwise transiently populated in the physiological environment are important questions to be addressed. These unexplored aspects are examined with villin headpiece (HP35) as a model protein confined within a mesopore of MIL-101(Cr) using molecular dynamics simulations. At equilibrium, the protein is located farther from the center of the cavity and closer to the MOF surface. Molecular interactions with the MOF partially unfold helix-1 at its N-terminus. Umbrella sampling simulations inform the range of conformations that HP35 undertakes during translocation from one cavity to another and associated changes in free energy. Relative to its equilibrium state within the cavity, the free energy barrier for the unfolded protein at the cage window is estimated to be 16 kcal/mol. This study of MOF-based protein conformation can also be a general approach to observing intermediates in folding-unfolding pathways., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Cells at disease onset are often associated with subtle changes in the expression level of a single or few molecular components, making traditionally used biomarker-driven clinical diagnosis a challenging task. We demonstrate here the design of a DNA nanosensor array with multichannel output that identifies the normal or pathological state of a cell based on the alteration of its global proteomic signature. Fluorophore-encoded single-stranded DNA (ssDNA) strands were coupled via supramolecular interaction with a surface-functionalized gold nanoparticle quencher to generate this integrated sensor array. In this design, ssDNA sequences exhibit dual roles, where they provide differential affinities with the receptor gold nanoparticle as well as act as transducer elements. The unique interaction mode of the analyte molecules disrupts the noncovalent supramolecular complexation, generating simultaneous multichannel fluorescence output to enable signature-based analyte identification via a linear discriminant analysis-based machine learning algorithm. Different cell types, particularly normal and cancerous cells, were effectively distinguished using their fluorescent fingerprints. Additionally, this DNA sensor array displayed excellent sensitivity to identify cellular alterations associated with chemical modulation of catabolic processes. Importantly, pharmacological effectors, which could modulate autophagic flux, have been effectively distinguished by generating responses from their global protein signatures. Taken together, these studies demonstrate that our multichannel DNA nanosensor is well suited for rapid identification of subtle changes in a complex mixture and thus can be readily expanded for point-of-care clinical diagnosis, high-throughput drug screening, or predicting the therapeutic outcome from a limited sample volume.