Lipid-Raft-Targeted Molecular Self-Assembly Inactivates YAP to Treat Ovarian Cancer.

The Yes-associated protein (YAP) is a major oncoprotein responsible for cell proliferation control. YAP's oncogenic activity is regulated by both the Hippo kinase cascade and uniquely by a mechanical-force-induced actin remodeling process. Inspired by reports that ovarian cancer cells specifically accumulate the phosphatase protein ALPP on lipid rafts that physically link to actin cytoskeleton, we developed a molecular self-assembly (MSA) technology that selectively halts cancer cell proliferation by inactivating YAP. We designed a ruthenium-complex-peptide precursor molecule that, upon cleavage of phosphate groups, undergoes self-assembly to form nanostructures specifically on lipid rafts of ovarian cancer cells. The MSAs exert potent, cancer-cell-specific antiproliferative effects in multiple cancer cell lines and in mouse xenograft tumor models. Our work illustrates how basic biochemical insights can be exploited as the basis for a nanobiointerface fabrication technology which links nanoscale protein activities at specific subcellular locations to molecular biological activities to suppress cancer cell proliferation.

Direct observation of adsorption-induced inactivation of antibody fragments surrounded by mixed-PEG layer on a gold surface.

To examine the adsorption behavior of antibody fragments (Fab') directly immobilized on a gold surface through S-Au linkage, analyses by surface plasmon resonance (SPR), fluorometry, and atomic force microscopy (AFM) with an excellent blocking technique by the consecutive treatments of longer-poly(ethylene glycol) (PEG) (MW = 5k) and shorter-PEG (MW = 2k), abbreviated as mixed-PEG layer formation, were performed. The results of the SPR analysis suggest that the adsorption-induced inactivation of the antigen-binding activity of Fab' took place gradually on the gold surface, where the activity disappeared almost completely at 60 min after Fab' immobilization. In contrast, in the case of Fab' coimmobilized by the mixed-PEG layer, 70% of the initial antigen-binding activity of the Fab' was retained even 60 min after the construction of the hybrid surface. Using fluorescein-labeled Fab' (FL-Fab'), fluorescence measurement of the constructed surface was carried out. The fluorescence of the FL-Fab' without any blocking agent on the gold surface was gradually quenched and finally decreased to 40% of the initial intensity 60 min after Fab' immobilization. The decrease in the fluorescence intensity is considered to be caused by the change in the distance between the fluorophores labeled on the Fab' and the gold surface, due to the energy transfer from the fluorophores to the gold surface. In contrast, 75% of the initial intensity was observed on the Fab'/mixed-PEG coimmobilized surface. The results obtained from the SPR and fluorometric analyses correlated well with each other; thus, the surface-induced inactivation of the antigen-binding functionality was presumably due to the conformational and/or orientation change of Fab' on the gold surface. AFM studies provided direct information on the time-dependent decrease in the height of the immobilized Fab' on the gold surface. In contrast, the coimmobilization of densely packed mixed-PEG tethered chains around the Fab' on the gold surface suppressed the decrease in the height of Fab', presumably indicating that the conformational and/or orientation change of Fab' was suppressed by the coimmobilized mixed-PEG layer. The new findings obtained in this study are expected to be useful for the improvement of the antibody fragment method and, thus, for the construction of high-performance immuno-surfaces.

A new method of biosensing with 1 microl of Escherichia coli suspension using atomic force microscopy.

We developed a new method for detecting bacterial cells from 1-mul samples with atomic force microscopy (AFM). The use of a parafilm surface as a sample palette was effective for reacting small amounts of samples with an AFM probe. This was due to the parafilm's hydrophobic, semitransparent, and nonadhesive surface. In this way, all processes, such as the surface functionalization of a cantilever and the adhesion of Escherichia coli cells to a cantilever, were easily completed. In addition, we succeeded in detecting cell adsorption on the same AFM cantilever by both the drive mode and the thermal mode. The resonance frequency shift caused by cell adhesion was clearly detected by the two modes for the first time. Our data indicated the potential of applying AFM nanobiosensing to extremely small amounts of samples.