Direct Visualization of the Dynamic Process of Epsilon Toxin on Hemolysis.

Hemolysis is the process of rupturing erythrocytes (red blood cells) by forming nanopores on their membranes using hemolysins, which then impede membrane permeability. However, the self-assembly process before the state of transmembrane pores and underlying mechanisms of conformational change are not fully understood. In this work, theoretical and experimental evidence of the pre-pore morphology of Clostridium perfringens epsilon toxin (ETX), a typical hemolysin, is provided using in situ atomic force microscopy (AFM) complemented by molecular dynamics (MD) simulations to detect the conformational distribution of different states in Mica. The AFM suggests that the ETX pore is formed in two stages: ETX monomers first attach to the membrane and form a pre-pore in no special conditions required, which then undergo a conformational change to form a transmembrane pore at temperatures above the critical point in the presence of receptors. The authors' MD simulations reveal that initial nucleation occurs when specific amino acids adsorb to negatively charged mica cavities. This work fills the knowledge gap in understanding the early stage of hemolysis and the oligomerization of hemolysins. Moreover, the newly identified pre-pore of ETX holds promise as a candidate for nanopore applications.

Selective In Situ Analysis of Mature microRNAs in Extracellular Vesicles Using a DNA Cage-Based Thermophoretic Assay.

Mature microRNAs (miRNAs) in extracellular vesicles (EVs) are involved in different stages of cancer progression, yet it remains challenging to precisely detect mature miRNAs in EVs due to the presence of interfering RNAs (such as longer precursor miRNAs, pre-miRNAs) and the low abundance of tumor-associated miRNAs. By leveraging the size-selective ability of DNA cages and polyethylene glycol (PEG)-enhanced thermophoretic accumulation of EVs, we devised a DNA cage-based thermophoretic assay for highly sensitive, selective, and in situ detection of mature miRNAs in EVs with a low limit of detection (LoD) of 2.05 fM. Our assay can profile EV mature miRNAs directly in serum samples without the interference of pre-miRNAs and the need for ultracentrifugation. A clinical study showed that EV miR-21 or miR-155 had an overall accuracy of 90 % for discrimination between breast cancer patients and healthy donors, which outperformed conventional molecular probes detecting both mature miRNAs and pre-miRNAs. We envision that our assay can advance EV miRNA-based diagnosis of cancer.

Tetrahedral DNA Nanostructure with Interferon Stimulatory DNA Delivers Highly Potent Toxins and Activates the cGAS-STING Pathway for Robust Chemotherapy and Immunotherapy.

Tumor metastases and reoccurrences are considered the leading cause of cancer-associated deaths. While highly efficient treatments for the eradication of primary tumors have been developed, the treatment of secondary or metastatic tumors remains poorly accessible. Over the past years, compounds that intervene through the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway against tumor metastases have emerged with potential for clinical development. While interferon stimulatory DNAs have demonstrated activation of this pathway, these compounds are associated with poor bioavailability, poor stability, and poor cancer selectivity, hindering their use for therapeutic applications. Herein, the encapsulation of a highly potent chemotherapeutic platinum(II) complex and the incorporation of interferon stimulatory DNA strands for activation of the cGAS-STING pathway into multimodal tetrahedral DNA nanostructures (84bp-TDNISD/56MESS ) for combined chemotherapy and immunotherapy is reported. It is found that 84bp-TDNISD/56MESS can work as not only a drug delivery carrier for highly potent toxins, but also an immunostimulant agent that can activate the STING pathway for antitumor immune responses. In a mouse breast cancer model, the DNA nanostructure is found to nearly fully eradicate primary as well as secondary/metastatic tumors, hence demonstrating its potential clinical translational value.

Spatiotemporal Control of Molecular Cascade Reactions by a Reconfigurable DNA Origami Domino Array.

Inspired by efficient biomolecular reactions in the cell, versatile DNA nanostructures have been explored for manipulating the spatial position and regulating reactions at the molecular level. Spatially controlled arrangement of molecules on the artificial scaffolds generally leads to enhanced reaction activities. Especially, the rich toolset of dynamic DNA nanostructures provides a potential route towards more sophisticated and vigorous regulation of molecular reactions. Herein, a reconfigurable DNA origami domino array (DODA) as a dynamic scaffold was adopted in this work for temporal-controlled and switchable molecular cascade reactions. Dynamic regulation of the assembly of G-quadruplex, hybridization of parallel-stranded duplex and assembly of binary DNAzyme were demonstrated. Molecular cascade reactions on the triggered reconfiguration of DODAs were realized, resulting in more complex, dynamic, and switchable control over the reactions.