Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

Tumor suppressor p53 plays a central role in response to DNA damage. DNA-damaging agents modulate nuclear actin dynamics, influencing cell behaviors; however, whether p53 affects the formation of nuclear actin filaments remains unclear. In this study, we found that p53 depletion promoted the formation of nuclear actin filaments in response to DNA-damaging agents, such as doxorubicin (DOXO) and etoposide (VP16). Even though the genetic probes used for the detection of nuclear actin filaments exerted a promotive effect on actin polymerization, the detected formation of nuclear actin filaments was highly dependent on both p53 depletion and DNA damage. Whilst active p53 is known to promote caspase-1 expression, the overexpression of caspase-1 reduced DNA damage-induced formation of nuclear actin filaments in p53-depleted cells. In contrast, co-treatment with DOXO and the pan-caspase inhibitor Q-VD-OPh or the caspase-1 inhibitor Z-YVAD-FMK induced the formation of nuclear actin filament formation even in cells bearing wild-type p53. These results suggest that the p53-caspase-1 axis suppresses DNA damage-induced formation of nuclear actin filaments. In addition, we found that the expression of nLifeact-GFP, the filamentous-actin-binding peptide Lifeact fused with the nuclear localization signal (NLS) and GFP, modulated the structure of nuclear actin filaments to be phalloidin-stainable in p53-depleted cells treated with the DNA-damaging agent, altering the chromatin structure and reducing the transcriptional activity. The level of phosphorylated H2AX (γH2AX), a marker of DNA damage, in these cells also reduced upon nLifeact-GFP expression, whilst details of the functional relationship between the formation of nLifeact-GFP-decorated nuclear actin filaments and DNA repair remained to be elucidated. Considering that the loss of p53 is associated with cancer progression, the results of this study raise a possibility that the artificial reinforcement of nuclear actin filaments by nLifeact-GFP may enhance the cytotoxic effect of DNA-damaging agents in aggressive cancer cells through a reduction in gene transcription.

The iron chelator deferriferrichrysin induces paraptosis via extracellular signal-related kinase activation in cancer cells.

Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF-7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal-related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy-treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK-dependent paraptosis.

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.

Simple and fast screening for structure-selective G-quadruplex ligands.

Structural selectivity of G-quadruplex ligands is essential for cellular applications since there is an excess of nucleic acids forming duplex structures compared to G-quadruplex structures in living cells. In this study, we developed new structure-selective G-quadruplex ligands utilizing a simple and fast screening system. The affinity, selectivity, enzymatic inhibitory activity and cytotoxicity of the structure-selective G-quadruplex ligands were demonstrated along with a structural selectivity-cytotoxicity relationship of G-quadruplex ligands.

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.

Significant structural change in human c-Myc promoter G-quadruplex upon peptide binding in potassium.

We selected the G-quadruplex motif located in the nuclease-hypersensitive elements (NHE) III1 region of the c-Myc promoter and for the first time performed its interaction studies with a designed peptide (QW10). Our CD results showed that the peptide bound to the c-Myc G-quadruplex and induced a significant blue shift in the positive peak of 20 nm in KCl alone or with 40wt% PEG200 or 20wt% PEG8000 in comparison to NaCl. Our Native Gel results confirmed that peptide binding destabilized the duplex and stabilized the unimolecular G-quadruplex and not binding to i-motif. UV thermal results confirmed destabilization of bimolecular structure and stabilization of unimolecular G-quadruplex. QW10 showed preferential binding towards c-MYC promoter G4 with binding constant (K b) values of the order of 0.05 ± 0.2 µM, 0.12 ± 0.1 µM and 0.05 ± 0.3 µM for complexes in K+ alone or 40wt% PEG 200 or 20wt% PEG 8000 respectively. QW10 showed preferential cytotoxicity with IC50 values of 11.10 µM and 6.44 µM after 72 and 96 hours' incubation on Human Breast Carcinoma MDA-MB 231 cells and was found to be non-toxic with Human Embryonic Kidney (HEK-1) cells. Interestingly, we observed reduction of c-Myc gene expression by 2.5 fold due to QW10 binding and stabilizing c-MYC G4. Our study for the first time provides an expanded overview of significant structural change in human c-Myc promoter G-quadruplex upon peptide binding in potassium.

Intramolecular G-quadruplex-hairpin loop structure competition of a GC-rich exon region in the TMPRSS2 gene.

We identified cytosine-rich regions adjacent to guanine-rich regions in protease genes. A typical GC-rich sequence derived from the TMPRSS2 gene showed structural competition between a G-quadruplex and a hairpin loop, and this competition significantly affected transcription efficiency. These results suggest an impact of neighboring sequences on the gene expression of guanine-rich sequences.

Detection of Intracellular Reactive Oxidative Species Using the Fluorescent Probe Hydroxyphenyl Fluorescein.

Various fluorescent probes for the detection of intracellular reactive oxidative species (ROS) have been developed because ROS levels are closely associated with cellular states. Here, we describe a method for detection of intracellular ROS in living cells using the fluorescent probe, hydroxyphenyl fluorescein (HPF), which detects hydroxyl radicals and peroxynitrite. NIH3T3 cells and p53 knockout (p53-/-) mouse embryonic fibroblasts (MEFs) were transformed by expressing oncogenic RAS using a retrovirus system. The cells were treated with HPF at 37 °C for 30 min, and subsequently, images were acquired using a confocal fluorescence microscope at an excitation wavelength of 488 nm after washing with PBS.

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.

Artificial turn-on riboswitch to control target gene expression using a wild-type riboswitch splicing mechanism.

The thiamine pyrophospate (TPP)-dependent thiA riboswitch in Aspergillus oryzae regulates alternative mRNA splicing via TPP-riboswitch binding to reduce protein production. Based on the sequences involved in alternative splicing found in Neurospora crassa, we identified unique sequences that are thought to play a role in the alternative splicing of the thiA riboswitch. Based on the mechanism of alternative splicing regulated by the thiA riboswitch, we constructed a new TPP-dependent artificial riboswitch, which unlike the wild-type riboswitch, promotes, rather than inhibits, gene expression. We found that a target gene controlled by this turn-on artificial riboswitch can be sufficiently expressed for practical use in A. oryzae. The artificial riboswitch upregulates the target genes via TPP and can be applied as a practical tool for gene regulation.

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).

RNA phase separation-mediated direction of molecular trafficking under conditions of molecular crowding.

Living cells are highly crowded with large and small biomolecules. The total concentration of biomolecules can reach 400 mg/ml, and 40% of the cell volume is occupied by biomolecules. Droplet formation in cells via liquid-liquid phase separation may play a role in controlling biochemical reactions in this complex molecular environment. Liquid-liquid phase separation generally involves nucleic acids and proteins as anionic and cationic components, respectively. Significant characteristics of droplets, which make them different from protein aggregation or fibril formation, are reversibility of formation and responsiveness to the molecular environment. In this review, we quantitatively describe the molecular environment inside cells and droplets that participate in controlling central dogma reactions. Finally, we discuss the importance of droplets under conditions of molecular crowding within living cells.

Hydroxyl groups in cosolutes regulate the G-quadruplex topology of telomeric DNA.

Telomeric G-quadruplex topology has the ability to regulate telomerase activity, which counteracts the shortening of telomere with successive cell divisions, thereby causing genomic longevity. However, the detailed mechanism of G-quadruplexes topologies formed by telomeric sequences requires further investigation. In this study, we quantitatively investigated the effect of cosolutes, particularly the varying number of hydroxyl groups, on the structural transition between hybrid type and parallel G-quadruplexes formed by telomeric DNA sequences. Cosolutes with one or no hydroxyl groups in the vicinal position more efficiently induced the transition to parallel G-quadruplex from hybrid G-quadruplex than those with more hydroxyl groups. We also examined the effect of cosolute structures on the hydration of G-quadruplex formation; the results indicated that cosolutes with fewer hydroxyl groups lead to the release of greater amount of water during G-quadruplex formation. Molecular dynamics results showed that the parallel G-quadruplex was more dehydrated than the hybrid type G-quadruplex. Generally, a dehydrated structure is favored under crowding condition. Thus, depending on the surrounding cosolutes, the G-quadruplex topology can be controlled by the G-quadruplex hydration state.

Osmolyte-Enhanced Protein Synthesis Activity of a Reconstituted Translation System.

Molecular crowding is receiving great attention in cell-free synthetic biology because molecular crowding is a critical feature of natural cell discrimination from artificial cells. Further, it has significant and generic influences on biomolecular functions. Although there are reports on how the macromolecular crowder reagents affect cell-free systems such as transcription and translation, the second class of molecular crowder reagents with low molecular weight, osmolyte, was much less studied in cell-free systems. In the present study, we focused on trimethylamine- N-oxide (TMAO) and betaine, methylamine osmolytes, and investigated the effectiveness of these osmolytes on gene expression activity of reconstituted cell-free protein synthesis. The gene expression activity of the fluorescent proteins Venus and tdTomato and the enzymes ß-galactosidase and dihydrofolate reductase were tested. At 37 °C, 0.4 M TMAO showed the highest enhancement of translational activity by a factor of 1.6-3.8, regardless of protein type. In contrast, betaine showed only a moderate effect that was limited to fluorescent proteins. Excess amounts of osmolytes suppressed gene expression activity. An mRNA-start assay and SDS-PAGE quantitative analysis provided firm evidence that TMAO enhances the translation process, instead of transcription, folding, or the maturation of fluorescent proteins. Interestingly, at 26 °C, TMAO and betaine showed the highest enhancement of protein synthesis activity at lower concentrations than at 37 °C. These findings provide implications on how osmolytes assist translation in natural cells. Further, they provide guidelines for modulation of protein synthesis activity in artificial cells through osmolyte addition.

Metal sensitive and DNA concentration dependent structural rearrangement of short oligonucleotide into large suprastructures.

Formation of higher order structures, such as G-quadruplexes and G-quadruplex based large suprastructures into long G-wires and liquid crystals is promising elements for use in healthcare for drug delivery as they are mechanically and thermally stable. In this study, we studied the structures of short 11-mer oligonucleotide 5'-G2AG5AG2-3'(11Pu) which is observed in 3'-UTR region of c-jun protooncogene. We used circular dichroism, UV-thermal melting, native gel electrophoresis and atomic force microscopy to determine the structure of 11Pu. CD results showed that 11Pu formed a mixed G-quadruplex in the presence of Na+ with and without Mg2+, while it formed a parallel G-quadruplex in the presence of 100 mM K+ with or without Mg2+. Cation selectivity in inducing the formation of large superstructures was observed in the presence of 100 mM K+ with 10 mM Mg2+. On the contrary, 10 mM Ca2+ did not induce the suprastructures. It was further demonstrated that Mg2+ at low concentration induced a parallel G-quadruplex of 11Pu, whereas at 10 mM Mg2+ induced a large suprastructure. AFM Images showed that 11Pu formed a G-wire, a liquid crystals and a crystalline lattice depending on the concentration of 11Pu and Mg2+. These insights may be employed to design G quadruplex-based nanowires for targeted drug delivery as well as interesting candidates for molecular nanowires. Communicated by Ramaswamy H. Sarma.

Selective recognition of human telomeric G-quadruplex with designed peptide via hydrogen bonding followed by base stacking interactions.

We described a novel synthetic peptide in which a glutamine residue binds through hydrogen bonding to a guanine-base and a trytophan residue intercalates with K+ resulting in stabilization of a human telomeric G-quadruplex with high selectivity over its complementary c-rich strand and a double-stranded DNA and its complementary C-rich strand. This peptide offers great potential for cancer treatment by inhibiting the telomere extension by telomerase.

An anionic phthalocyanine decreases NRAS expression by breaking down its RNA G-quadruplex.

Aberrant activation of RAS signalling pathways contributes to aggressive phenotypes of cancer cells. The RAS-targeted therapies for cancer, therefore, have been recognised to be effective; however, current developments on targeting RAS have not advanced due to structural features of the RAS protein. Here, we show that expression of NRAS, a major isoform of RAS, can be controlled by photo-irradiation with an anionic phthalocyanine, ZnAPC, targeting NRAS mRNA. In vitro experiments reveal that ZnAPC binds to a G-quadruplex-forming oligonucleotide derived from the 5'-untranslated region of NRAS mRNA even in the presence of excess double-stranded RNA, which is abundant in cells, resulting in selective cleavage of the target RNA's G-quadruplex upon photo-irradiation. In line with these results, upon photo-irradiation, ZnAPC decreases NRAS mRNA and NRAS expression and thus viability of cancer cells. These results indicate that ZnAPC may be a prominent photosensitiser for a molecularly targeted photodynamic therapy for cancer.

DNA G-Wire Formation Using an Artificial Peptide is Controlled by Protease Activity.

The development of a switching system for guanine nanowire (G-wire) formation by external signals is important for nanobiotechnological applications. Here, we demonstrate a DNA nanostructural switch (G-wire <--> particles) using a designed peptide and a protease. The peptide consists of a PNA sequence for inducing DNA to form DNA-PNA hybrid G-quadruplex structures, and a protease substrate sequence acting as a switching module that is dependent on the activity of a particular protease. Micro-scale analyses via TEM and AFM showed that G-rich DNA alone forms G-wires in the presence of Ca2+, and that the peptide disrupted this formation, resulting in the formation of particles. The addition of the protease and digestion of the peptide regenerated the G-wires. Macro-scale analyses by DLS, zeta potential, CD, and gel filtration were in agreement with the microscopic observations. These results imply that the secondary structure change (DNA G-quadruplex <--> DNA/PNA hybrid structure) induces a change in the well-formed nanostructure (G-wire <--> particles). Our findings demonstrate a control system for forming DNA G-wire structures dependent on protease activity using designed peptides. Such systems hold promise for regulating the formation of nanowire for various applications, including electronic circuits for use in nanobiotechnologies.

Highly Sensitive Telomerase Assay Insusceptible to Telomerase and Polymerase Chain Reaction Inhibitors for Cervical Cancer Screening Using Scraped Cells.

A sensitive telomerase assay based on asymmetric-polymerase chain reaction (A-PCR) on magnetic beads and subsequent application of cycling probe technology, STAMC, which is insusceptible to DNase and PCR inhibitors, was for the first time applied to clinical specimens in addition to a conventional telomeric repetitive amplification protocol (TRAP). The electrophoresis results showed that an increase in scraped cervical cancer cells not only reduced TRAP products but also increased smaller products, suggesting the unreliability of TRAP for clinical samples. To achieve the required sensitivity of STAMC for clinical application, the sequence and concentration conditions were explored for the forward and reverse primers for A-PCR, which resulted in a detection limit of only two HeLa cells with 1 µM TS primer (5'-AATCCGTCGAGCAGAGTT-3') and 0.04 µM ACX primer (5'-GCGCGGCTTACCCTTACCCTTACCCTAACC-3'). Under the same primer conditions, the fluorescence signal of STAMC increased as scraped cervical cancer cells increased despite showing a negligible intensity for benign tumors. Furthermore, STAMC showed no signal for a cervical cancer patient treated with irradiation therapy. These results indicate that STAMC is useful for not only cervical cancer screening but also investigating the effect of cancer treatments such as radiation therapy and drug administration.

Unexpected Position-Dependent Effects of Ribose G-Quartets in G-Quadruplexes.

To understand the role of ribose G-quartets and how they affect the properties of G-quadruplex structures, we studied three systems in which one, two, three, or four deoxyribose G-quartets were substituted with ribose G-quartets. These systems were a parallel DNA intramolecular G-quadruplex, d(TTGGGTGGGTTGGGTGGGTT), and two tetramolecular G-quadruplexes, d(TGGGT) and d(TGGGGT). Thermal denaturation experiments revealed that ribose G-quartets have position-dependent and cumulative effects on G-quadruplex stability. An unexpected destabilization was observed when rG quartets were presented at the 5'-end of the G stack. This observation challenges the general belief that RNA residues stabilize G-quadruplexes. Furthermore, in contrast to past proposals, hydration is not the main factor determining the stability of our RNA/DNA chimeric G-quadruplexes. Interestingly, the presence of rG residues in a central G-quartet facilitated the formation of additional tetramolecular G-quadruplex topologies showing positive circular dichroism signals at 295 nm. 2D NMR analysis of the tetramolecular TGgGGT (lowercase letter indicates ribose) indicates that Gs in the 5'-most G-quartet adopt the syn conformation. These analyses highlight several new aspects of the role of ribose G-quartets on G-quadruplex structure and stability, and demonstrate that the positions of ribose residues are critical for tuning G-quadruplex properties.