Combined Effects of Methylated Cytosine and Molecular Crowding on the Thermodynamic Stability of DNA Duplexes.

Methylated cytosine within CpG dinucleotides is a key factor for epigenetic gene regulation. It has been revealed that methylated cytosine decreases DNA backbone flexibility and increases the thermal stability of DNA. Although the molecular environment is an important factor for the structure, thermodynamics, and function of biomolecules, there are few reports on the effects of methylated cytosine under a cell-mimicking molecular environment. Here, we systematically investigated the effects of methylated cytosine on the thermodynamics of DNA duplexes under molecular crowding conditions, which is a critical difference between the molecular environment in cells and test tubes. Thermodynamic parameters quantitatively demonstrated that the methylation effect and molecular crowding effect on DNA duplexes are independent and additive, in which the degree of the stabilization is the sum of the methylation effect and molecular crowding effect. Furthermore, the effects of methylation and molecular crowding correlate with the hydration states of DNA duplexes. The stabilization effect of methylation was due to the favorable enthalpic contribution, suggesting that direct interactions of the methyl group with adjacent bases and adjacent methyl groups play a role in determining the flexibility and thermodynamics of DNA duplexes. These results are useful to predict the properties of DNA duplexes with methylation in cell-mimicking conditions.

Biomolecular Liquid-Liquid Phase Separation for Biotechnology.

The liquid-liquid phase separation (LLPS) of biomolecules induces condensed assemblies called liquid droplets or membrane-less organelles. In contrast to organelles with lipid membrane barriers, the liquid droplets induced by LLPS do not have distinct barriers (lipid bilayer). Biomolecular LLPS in cells has attracted considerable attention in broad research fields from cellular biology to soft matter physics. The physical and chemical properties of LLPS exert a variety of functions in living cells: activating and deactivating biomolecules involving enzymes; controlling the localization, condensation, and concentration of biomolecules; the filtration and purification of biomolecules; and sensing environmental factors for fast, adaptive, and reversible responses. The versatility of LLPS plays an essential role in various biological processes, such as controlling the central dogma and the onset mechanism of pathological diseases. Moreover, biomolecular LLPS could be critical for developing new biotechnologies such as the condensation, purification, and activation of a series of biomolecules. In this review article, we introduce some fundamental aspects and recent progress of biomolecular LLPS in living cells and test tubes. Then, we discuss applications of biomolecular LLPS toward biotechnologies.

Controlling liquid-liquid phase separation of G-quadruplex-forming RNAs in a sequence-specific manner.

We constructed a minimum liquid-liquid phase separation model system to form liquid droplets using only G-quadruplex-forming oligonucleotides and R- and G-rich oligopeptides. We found that the G-quadruplex structure is an essential component for RNA to form droplets with the peptide. Based on this model system and our findings, droplet redissolution via structure transition from a G-quadruplex to a duplex was achieved in a sequence-specific manner.

Photosensitizers Based on G-Quadruplex Ligand for Cancer Photodynamic Therapy.

G-quadruplex (G4) is the non-canonical secondary structure of DNA and RNA formed by guanine-rich sequences. G4-forming sequences are abundantly located in telomeric regions and in the promoter and untranslated regions (UTR) of cancer-related genes, such as RAS and MYC. Extensive research has suggested that G4 is a potential molecular target for cancer therapy. Here, we reviewed G4 ligands as photosensitizers for cancer photodynamic therapy (PDT), which is a minimally invasive therapeutic approach. The photosensitizers, such as porphyrins, were found to be highly toxic against cancer cells via the generation of reactive oxidative species (ROS) upon photo-irradiation. Several porphyrin derivatives and analogs, such as phthalocyanines, which can generate ROS upon photo-irradiation, have been reported to act as G4 ligands. Therefore, they have been implicated as promising photosensitizers that can selectively break down cancer-related DNA and RNA forming G4. In this review, we majorly focused on the potential application of G4 ligands as photosensitizers, which would provide a novel strategy for PDT, especially molecularly targeted PDT (mtPDT).