Engineered Extracellular Vesicles with Compound-Induced Cargo Delivery to Solid Tumors.

Efficient delivery of functional factors into target cells remains challenging. Although extracellular vesicles (EVs) are considered to be potential therapeutic delivery vehicles, a variety of efficient therapeutic delivery tools are still needed for cancer cells. Herein, we demonstrated a promising method to deliver EVs to refractory cancer cells via a small molecule-induced trafficking system. We generated an inducible interaction system between the FKBP12-rapamycin-binding protein (FRB) domain and FK506 binding protein (FKBP) to deliver specific cargo to EVs. CD9, an abundant protein in EVs, was fused to the FRB domain, and the specific cargo to be delivered was linked to FKBP. Rapamycin recruited validated cargo to EVs through protein-protein interactions (PPIs), such as the FKBP-FRB interaction system. The released EVs were functionally delivered to refractory cancer cells, triple negative breast cancer cells, non-small cell lung cancer cells, and pancreatic cancer cells. Therefore, the functional delivery system driven by reversible PPIs may provide new possibilities for a therapeutic cure against refractory cancers.

Platform- and label-free detection of lead ions in environmental and laboratory samples using G-quadraplex probes by circular dichroism spectroscopy.

Guanine-rich quadruplex (G-QD) are formed by conversion of nucleotides with specific sequences by stabilization of positively charged K+ or Na+. These G-QD structures differentially absorb two-directional (right- and left-handed) circularly polarized light, which can discriminate the parallel or anti-parallel structures of G-QDs. In this study, G-QDs stabilized by Pb2+ were analyzed by a circular dichroism (CD) spectroscopy to determine Pb2+ concentration in water samples. Thrombin aptamer (TBA), PS2.M, human telomeric DNA (HTG), AGRO 100, and telomeric related sequence (T2) were studied to verify their applicability as probes for platform- and label-free detection of Pb2+ in environmental as well as laboratory samples. Among these nucleotides, TBA and PS2.M exhibited higher binding constants for Pb2+, 1.20-2.04 × 106/M at and 4.58 × 104-1.09 × 105/M at 100 micromolar and 100 mM K+ concentration, respectively. They also exhibited excellent selectivity for Pb2+ than for Al3+, Cu2+, Ni2+, Fe3+, Co2+, and Cr2+. When Pb2+ was spiked into an effluent sample from a wastewater treatment plant (WWTP), its existence was detected by CD spectroscopy following a simple addition of TBA or PS2.M. By the addition of TBA and PS2.M, the Pb2+ signals were observed in effluent samples over 0.5 micromolar (100 ppb) concentration. Furthermore, PS2.M caused a Pb2+-specific absorption band in the effluent sample without spiking of Pb2+, and could be induced to G-QD structure by the background Pb2+ concentration in the effluent, 0.159 micromolar concentration (3.30 ppb). Taken together, we propose that TBA and PS2.M are applicable as platform- and label-free detection probes for monitoring Pb2+ in environmental samples such as discharged effluent from local WWTPs, using CD spectroscopy.

RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro.

Mg2+ ion stimulates the DNA strand exchange reaction catalyzed by RecA, a key step in homologous recombination. To elucidate the molecular mechanisms underlying the role of Mg2+ and the strand exchange reaction itself, we investigated the interaction of RecA with Mg2+ and sought to determine which step of the reaction is affected. Thermal stability, intrinsic fluorescence, and native mass spectrometric analyses of RecA revealed that RecA binds at least two Mg2+ ions with KD ≈ 2 mM and 5 mM. Deletion of the C-terminal acidic tail of RecA made its thermal stability and fluorescence characteristics insensitive to Mg2+ and similar to those of full-length RecA in the presence of saturating Mg2+. These observations, together with the results of a molecular dynamics simulation, support the idea that the acidic tail hampers the strand exchange reaction by interacting with other parts of RecA, and that binding of Mg2+ to the tail prevents these interactions and releases RecA from inhibition. We observed that binding of the first Mg2+ stimulated joint molecule formation, whereas binding of the second stimulated progression of the reaction. Thus, RecA is actively involved in the strand exchange step as well as bringing the two DNAs close to each other.