Observation and Analysis of Model Lipid Raft Deformation Dynamics Induced by Lipoprotein–Gold Nanorod-based Devices to Manipulate Phase Transition of Lipid Bilayer

Lipid rafts, which are complexes that form on cholesterol-rich areas of cell membranes, play key roles as gates for the distribution of information between the inner and outer spaces of living cells. The physical and biological properties of lipid rafts have been investigated, but the engineering of lipid rafts remains a difficult subject. We developed a lipoprotein–gold nanorod nanodevice to control the formation/deformation of model lipid rafts. The nanodevice attached to the liquid order (Lo) phase region, selectively removed cholesterol from the Lo domain, and induced the Lo-to-solid order (So) phase transition. In this study, we analyzed the phase transition induced by the nanodevice. We found that the domain boundary gradually collapsed and that a local two-phase mixture was induced. Local instability resulted in domain incursion into other domains, and protrusions were torn to form small domains with the characteristics of two phases. The two different domains grouped together to form a non-circular So domain on giant unilamellar vesicles. Close observation of the non-equilibrium process of phase transition may lead to the design of strategies for external lipid raft manipulation methodology using biological macromolecules and/or nanoparticles

Phase-diagram observation of liquid–liquid phase separation in the poly-L-lysine/ATP system and diagram-based application strategy

Liquid–liquid phase separation (LLPS) is essential to understand biomacromolecule compartmentalization in living cells and to form soft-matter structures for chemical reactions and drug delivery systems. However, the importance of detail experimental phase diagrams of modern LLPS systems tend to be overlooked nowadays. Even for poly-L-lysine (PLL)/ATP system, one of the most widely used LLPS models, detailed phase diagram of LLPS have not been obtained. Herein, we report the first phase diagram of the (PLL)/ATP system and demonstrate the feasibility of phase-diagram based research design not only for understanding the physical properties of LLPS systems but also for realizing biophysical and medical applications. We established an experimental handy model of the droplet formation/disappearance process by forming a concentration gradient in a chamber as extracting a suitable condition on the phase-diagram including the two-phase droplet region. As a proof of concept of pharmaceutical application, we added a human immunoglobulin G (IgG) solution to PLL/ATP system. Using the knowledge of the phase diagram, we enabled to form IgG/PLL droplets clearly in the pharmaceutically required IgG concentration of ca. 10 mg/mL. This study provides a guidance for using the phase diagram to analyze and utilize LLP

Interfacial and intrinsic molecular effects on the phase separation/transition of heteroprotein condensates

Liquid–liquid phase separation (LLPS) and droplet formation by LLPS are key concepts used to explain compartmentalization in living cells. Protein contact to a membrane surface is considered an important process for protein organization in a liquid phase or during transition to a solid or liquid dispersion state. The direct experimental comprehensive investigation is; however, not performed on the surface–droplet interaction and phase transition. In the present study, we constructed simple and reproducible experiments to analyze the structural transition of aggregates and droplets in an ovalbumin (OVA) and lysozyme (LYZ) complex on glass slides with various coatings. The difference in droplet-surface interaction may only be important in the boundary region between aggregates and droplets of a protein mixture, as shown in the phase diagram. Co-aggregates of OVA-LYZ changed to droplet-like circular forms during incubation. In contrast, free L-lysine resulted in the uniform droplet-to-solid phase separation at lower concentrations and dissolved any structures at higher concentrations. These results represent the first phase-diagram-based analysis of the phase transition of droplets in a protein mixture and a comparison of surface-surface and small molecular-droplet structure interactions

Regulation of the Liquid–liquid Phase-Separated Droplets of Biomacromolecules by Butterfly-Shaped Gold Nanomaterials

Liquid–liquid phase-separated (LLPS) droplets play key roles in regulating protein behaviors, such as enzyme compart-mentalization, stress response, and disease pathogenesis, in living cells. The manipulation of the droplet for-mation/deformation dynamics is the next target of nano-biotechnology, although the required nanodevices for controlling the dynamics of liquid–liquid phase separation, LLPS, have not been invented. Here, we propose a butterfly-shaped gold nanobutterfly (GNB) as a nanodevice for manipulating the droplet-formation/deformation dynamics of LLPS. GNBs are moderate, symmetrical gold nanomaterials (average diameter = ~30 nm) bearing two concaves and resembling a butter-fly. Their growth process is analyzed via their time-lapse electroscopic images and time-lapse ultraviolet/visible/near-infrared (NIR) spectroscopy, as well as the application of solution additives in protein science. These nanomaterials are synthesized via the seed-mediated method with an efficiency of ~70%. Interestingly, the GNBs stabilized the LLPS droplet of adenosine triphosphate (ATP)/poly-L-lysine, whereas other two gold nanoparticles with different shapes (spherical and rod-shaped) did not, indicating that the concave of the GNBs interacts with the precursor of the droplets. The NIR-laser irradiation of the GNBs facilitates the on-demand deformation of the droplets via the localized-heat effect. This but-terfly-shaped nanodevice represents a future strategy for manipulating the dynamics of LLPS

Creation of a Synthetic Ligand for Mitochondrial DNA Sequence Recognition and Promoter-Specific Transcription Suppression

Synthetic ligands capable of recognizing the specific DNA sequences inside human mitochondria and modulating gene transcription are in increasing demand because of the surge in evidence linking mitochondrial genome and diseases. In the work described herein, we created a new type of mitochondria-specific synthetic ligand, termed MITO-PIPs, by conjugating a mitochondria-penetrating peptide with pyrrole-imidazole polyamides (PIPs). The designed MITO-PIPs showed specific localization inside mitochondria in HeLa cells and recognized the target DNA in a sequence-specific manner. Furthermore, MITO-PIPs that inhibit the binding of mitochondrial transcription factor A to the light-strand promoter (LSP) also triggered targeted transcriptional suppression. The tunability of PIPs’ properties suggests the potential of the MITO-PIPs as potent modulators of not only mitochondrial gene transcription but also its DNA mutations