Small molecule-based detection of non-canonical RNA G-quadruplex structures that modulate protein translation.

Tandem repeats of guanine-rich sequences in RNA often form thermodynamically stable four-stranded RNA structures. Such RNA G-quadruplexes have long been considered to be linked to essential biological processes, yet their physiological significance in cells remains unclear. Here, we report a approach that permits the detection of RNA G-quadruplex structures that modulate protein translation in mammalian cells. The approach combines antibody arrays and RGB-1, a small molecule that selectively stabilizes RNA G-quadruplex structures. Analysis of the protein and mRNA products of 84 cancer-related human genes identified Nectin-4 and CapG as G-quadruplex-controlled genes whose mRNAs harbor non-canonical G-quadruplex structures on their 5'UTR region. Further investigations revealed that the RNA G-quadruplex of CapG exhibits a structural polymorphism, suggesting a possible mechanism that ensures the translation repression in a KCl concentration range of 25-100 mM. The approach described in the present study sets the stage for further discoveries of RNA G-quadruplexes.

RNA-based cooperative protein labeling that permits direct monitoring of the intracellular concentration change of an endogenous protein.

Imaging the dynamics of proteins in living cells is a powerful means for understanding cellular functions at a deeper level. Here, we report a versatile method for spatiotemporal imaging of specific endogenous proteins in living mammalian cells. The method employs a bifunctional aptamer capable of selective protein recognition and fluorescent probe-binding, which is induced only when the aptamer specifically binds to its target protein. An aptamer for ß-actin protein preferentially recognizes its monomer forms over filamentous forms, resulting in selective G-actin staining in both fixed and living cells. Through actin-drug treatment, the method permitted direct monitoring of the intracellular concentration change of endogenous G-actin. This protein-labeling method, which is highly selective and non-covalent, provides rich insights into the study of spatiotemporal protein dynamics in living cells.

Cas9-guided haplotyping of three truncation variants in autosomal recessive disease.

An autosomal recessive disease is caused by biallelic loss-of-function mutations. However, when more than two disease-causing variants are found in a patient's gene, it is challenging to determine which two of the variants are responsible for the disease phenotype. Here, to decipher the pathogenic variants by precise haplotyping, we applied nanopore Cas9-targeted sequencing (nCATS) to three truncation COL7A1 variants detected in a patient with recessive dystrophic epidermolysis bullosa (EB). The distance between the most 5' and 3' variants was approximately 19 kb at the level of genomic DNA. nCATS successfully demonstrated that the most 5' and 3' variants were located in one allele while the variant in between was located in the other allele. Interestingly, the proband's mother, who was phenotypically intact, was heterozygous for the allele that harbored the two truncation variants, which could otherwise be misinterpreted as those of typical recessive dystrophic EB. Our study highlights the usefulness of nCATS as a tool to determine haplotypes of complicated genetic cases. Haplotyping of multiple variants in a gene can determine which variant should be therapeutically targeted when nucleotide-specific gene therapy is applied.

Association between pulmonary oxygen uptake on-kinetics and acute cardiovascular responses to resistance exercise in patients with coronary artery disease: a preliminary study.

[Purpose] This study aimed to examine whether pulmonary oxygen uptake on-kinetics at the onset of moderate-intensity exercise can predict acute cardiovascular responses to resistance exercise. [Participants and Methods] The association between pulmonary oxygen uptake on-kinetics and acute cardiovascular responses to a single resistance exercise session was investigated in seven patients with low-risk coronary artery disease who underwent revascularization through percutaneous coronary intervention. The participants performed a cardiopulmonary exercise test on a cycle ergometer and a single resistance exercise session at 30% of maximum voluntary contraction on a bilateral leg-extension machine 1 week after surgery. We measured the ventilatory anaerobic threshold and pulmonary oxygen uptake on-kinetics during the cardiopulmonary exercise test; left ventricular ejection fraction at rest; and heart rate, systolic blood pressure, and rate pressure product during the single resistance exercise session. [Results] Pulmonary oxygen uptake on-kinetics showed a positive association with the amount of increase in systolic blood pressure and rate pressure product during the single resistance exercise session, but had no association with the amount of increase in heart rate. Ventilatory anaerobic threshold and left ventricular ejection fraction were not associated with these parameters. [Conclusion] These data suggested that pulmonary oxygen uptake on-kinetics can be a useful evaluation index for predicting acute systolic blood pressure and rate pressure product responses to low-intensity resistance exercise 1 week after percutaneous coronary intervention in patients with low-risk coronary artery disease.

G-quadruplexes in mRNA: A key structure for biological function.

The last several years have seen exciting advances in the understanding of the structure and function of higher-order structures of RNA. Expression levels of some specific genes were shown to be directly regulated by environmentally-responsive formation of certain secondary structures such as stem-loops and pseudoknots. Even among these noncanonical structures, RNA G-quadruplexes, which form on the regions of guanine-rich sequences in mRNA, are highly stable structures that are involved in a variety of biological processes. However, many questions regarding the biological significance of RNA G-quadruplexes remain unsettled, mainly because it is difficult to locate the structures in mRNA. This review focuses on emerging methods that locate RNA G-quadruplexes in mRNA by computational and biochemical techniques. In addition, recent reports on the biological functions of RNA G-quadruplexes are also covered to highlight their various roles in cells, such as in regulating mRNA processing and translation.

Risk Profile and 1-Year Outcome of Newly Diagnosed Atrial Fibrillation in Japan　- Insights From GARFIELD-AF.

BACKGROUND: Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective non-interventional study of stroke prevention in patients with newly diagnosed non-valvular AF (NAVF) that is being conducted in 35 countries. Methods and Results: A total of 52,081 patients with a new diagnosis of NVAF were enrolled prospectively in GARFIELD-AF. Of these, 4859 (9.3%) were recruited in Japan (2010-2016). In cohort 1 (2010-2011), few patients were on non-vitamin K antagonist oral anticoagulants (NOAC) globally. From cohort 2 onwards (2011-2016), however, there was a rapid increase in NOAC use around the globe, especially in Japan. By the last year of enrolment (2015-2016), 67.9% of patients in Japan and 43.1% of patients globally were on NOAC±antiplatelet therapy (AP). In Japan and globally, 17.0% and 12.2% of patients, respectively, did not receive stroke prevention treatment. Few patients in Japan (5.7%) received AP only. Compared with the other countries, the unadjusted rates of all-cause mortality and major bleeding were low, while rates of stroke/systemic embolism were similar after 1 year of follow-up. CONCLUSIONS: GARFIELD-AF continues to provide important information on the homogeneity and heterogeneity of baseline characteristics and treatment patterns in patients with newly diagnosed NVAF. This diversity reflects the differences in outcomes in Japan compared with the rest of the world.

A Synthetic Hybrid Molecule for the Selective Removal of Human Pluripotent Stem Cells from Cell Mixtures.

A major hurdle in stem cell therapy is the tumorigenic risk of residual undifferentiated stem cells. This report describes the design and evaluation of synthetic hybrid molecules that efficiently reduce the number of human induced pluripotent stem cells (hiPSCs) in cell mixtures. The design takes advantage of Kyoto probe 1 (KP-1), a fluorescent chemical probe for hiPSCs, and clinically used anticancer drugs. Among the KP-1-drug conjugates we synthesized, we found an exceptionally selective, chemically tractable molecule that induced the death of hiPSCs. Mechanistic analysis suggested that the high selectivity originates from the synergistic combination of transporter-mediated efflux and the cytotoxicity mode of action. The present study offers a chemical and mechanistic rationale for designing selective, safe, and simple reagents for the preparation of non-tumorigenic clinical samples.

A Small Molecule That Represses Translation of G-Quadruplex-Containing mRNA.

The G-quadruplexes form highly stable nucleic acid structures, which are implicated in various biological processes in both DNA and RNA. Although DNA G-quadruplexes have been studied in great detail, biological roles of RNA G-quadruplexes have received less attention. Here, a screening of a chemical library permitted identification of a small-molecule tool that binds selectively to RNA G-quadruplex structures. The polyaromatic molecule, RGB-1, stabilizes RNA G-quadruplex, but not DNA versions or other RNA structures. RGB-1 intensified the G-quadruplex-mediated inhibition of RNA translation in mammalian cells, decreased expression of the NRAS proto-oncogene in breast cancer cells, and permitted identification of a novel sequence that forms G-quadruplex in NRAS mRNA. RGB-1 may serve as a unique tool for understanding cellular roles of RNA G-quadruplex structures.

Live-cell imaging of endogenous mRNAs with a small molecule.

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non-engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene-specific RNA aptamer, combined with a cell-permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live-cell imaging of the endogenous mRNA of ß-actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin-2, cortactin, and cytoplasmic FMR1-interacting protein 2. The RNA-imaging technology and its further optimization might permit live-cell imaging of any RNA molecules.

DNA origami based visualization system for studying site-specific recombination events.

Site-specific recombination involves reciprocal exchange between defined DNA sites. The reaction initiates from the formation of a recombinase-DNA synaptic complex, in which two recombination sites arrange in an appropriate configuration. However, there is incomplete information about how the topological state of the substrate influences the synapsis and outcome of the reaction. Here, we show that Cre-mediated recombination can be regulated by controlling the orientation and topology of the loxP substrate in a DNA frame nanoscaffold. High-speed atomic force microscopy analyses revealed that the loxP-containing substrate strands in the antiparallel orientation can be recombined only through formation of synaptic complexes. By tethering Holliday junction (HJ) intermediates to DNA frames in different connection patterns and using them as a starting substrate, we found that the topological state of the HJ intermediates dictates the outcome of the resolution. Our approach should provide a new platform for structural-functional studies of various DNA targeting enzymes, especially which require formation of synaptic complexes.

Metal Ion-Directed Specific DNA Structures and Their Functions.

Various DNA structures, including specific metal ion complexes, have been designed based on the knowledge of canonical base pairing as well as general coordination chemistry. The role of metal ions in these studies is quite broad and diverse. Metal ions can be targets themselves in analytical applications, essential building blocks of certain DNA structures that one wishes to construct, or they can be responsible for signal generation, such as luminescence or redox. Using DNA conjugates with metal chelators, one can more freely design DNA complexes with diverse structures and functions by following the simple HSAB rule. In this short review, the authors summarize a part of their DNA chemistries involving specific metal ion coordination. It consists of three topics: (1) significant stabilization of DNA triple helix by silver ion; (2) metal ion-directed dynamic sequence edition through global conformational change by intramolecular complexation; and (3) reconstruction of luminescent lanthanide complexes on DNA and their analytical applications.

Photo-cross-linking-assisted thermal stability of DNA origami structures and its application for higher-temperature self-assembly.

Heat tolerance of DNA origami structures has been improved about 30 °C by photo-cross-linking of 8-methoxypsoralen. To demonstrate one of its applications, the cross-linked origami were used for higher-temperature self-assembly, which markedly increased the yield of the assembled product when compared to the self-assembly of non-cross-linked origami at lower-temperature. By contrast, at higher-temperature annealing, native non-cross-linked tiles did not self-assemble to yield the desired product; however, they formed a nonspecific broken structure.

Catalytic Amplification of Electrochemical Signal in Homogeneous Solution Using an Entropy-driven DNA Circuit.

The electrochemical signal from ferrocene on a DNA probe was successfully modulated in a homogeneous solution by the template-directed formation and dissociation of an inclusion complex with ß-cyclodextrin on another probe. The electrochemical response was amplified by combining with a DNA circuit, in which the target DNA served as a catalyst. This system did not require any modification of a complementary DNA with the ferrocene-modified probe on the electrode surface to separate the bound/free probe for the detection of 200 nM target DNA.

The Effects of Long-Term Nutrition Counseling According to the Behavioral Modification Stages in Patients with Cardiovascular Disease.

BACKGROUND: the behavioral modification stages (BMS) are widely used; however, there are no reports on long-term nutrition counseling for cardiovascular disease (CVD) according to BMS. AIM: to study the effects of long-term nutrition counseling based on the BMS in patients with CVD. METHODS: fifteen patients with CVD who participated in nutrition counseling were enrolled between June 2012 and December 2016. We provided BMS and dietary questionnaires to estimate the stage score (SS), salt intake, and drinking habits (non-drinking group (n = 7)/drinking group (n = 8)), and measured the blood pressure (BP), body mass index (BMI), and biochemical markers before and after hospitalization at 6 months, 1 year, and 1.5 years after leaving the outpatient department (OPD). RESULTS: a significant decreased salt intake and increase in SS were found at 1.5 years. It significantly decreased the BP and salt intake in the non-drinking group at 1.5 years. CONCLUSIONS: long-term nutrition counseling according to BMS improved salt intake and BP in the non-drinking group. However, in the drinking group, increased salt intake might weaken the BP improvement. Temperance and low-sodium intake are essential factors that control BP, especially in drinkers.

Associations between High-Density Lipoprotein Functionality and Major Adverse Cardiovascular Events in Patients Who Have Undergone Coronary Computed Tomography Angiography.

The present study aimed to investigate the associations between high-density lipoprotein (HDL) functionality and major adverse cardiovascular events (MACE) in patients who have undergone coronary computed tomography angiography (CCTA). We performed a prospective cohort study and enrolled 151 patients who underwent CCTA and had a follow-up of up to 5 years. We measured cholesterol efflux capacity (CEC), caspase-3/7 activity and monocyte chemoattractant protein-1 (MCP-1) secretion as bioassays of HDL functionality. The patients were divided into MACE(-) (n = 138) and MACE(+) (n = 13) groups. While there was no significant difference in %CEC, caspase-3/7 activity or MCP-1 secretion between the MACE(-) and MACE(+) groups, total CEC and HDL cholesterol (HDL-C) in the MACE(+) group were significantly lower than those in the MACE(-) group. Total CEC was correlated with HDL-C. A receiver-operating characteristic curve analysis showed that there was no significant difference between the areas under the curves for total CEC and HDL-C. In conclusion, total CEC in addition to HDL-C, but not %CEC, was associated with the presence of MACE. On the other hand, HDL functionality with regard to anti-inflammatory and anti-apoptosis effects was not associated with MACE.

Association Between the Level of Low-Density Lipoprotein Cholesterol and Coronary Atherosclerosis in Patients Who Have Undergone Coronary Computed Tomography Angiography.

BACKGROUND: Although the Japan Atherosclerosis Society Guidelines 2017 recommend lower levels of low-density lipoprotein cholesterol (LDL-C, < 70 mg/dL or ≤ 100 mg/dL) to prevent secondary cardiovascular events, we cannot conclude that a low level of LDL-C prevents primary cardiovascular events in patients with suspected coronary artery disease (CAD). METHODS: We registered 1,016 patients who were clinically suspected to have CAD and who underwent coronary computed tomography angiography (CCTA) for screening of coronary atherosclerosis. We excluded 350 patients who were receiving anti-lipidemic therapies and finally analyzed 666 patients. The patients were divided into three groups according to the LDL-C level: < 70 mg/dL (n = 25, Low LDL-C), 70 - 99 mg/dL (n = 141, Middle LDL-C), and ≥ 100 mg/dL (n = 500, High LDL-C). A ≥ 50% coronary stenosis was initially diagnosed as CAD, and the number of significantly stenosed coronary vessels (VD), Gensini score and coronary artery calcification (CAC) score were quantified. RESULTS: There were no significant differences in age, high-density lipoprotein cholesterol, rates of hypertension, hemoglobin A1c, blood sugar or systolic blood pressure among the Low, Middle and High LDL-C groups. On the other hand, there were significant differences in rates of males, smoking, dyslipidemia and diabetes, diastolic blood pressure and triglyceride among the groups. The prevalence of CAD values in the Low, Middle and High LDL-C groups were similar, at 52%, 47%, and 46%, respectively. In addition, there were no significant differences in the number of VD, Gensini score or CAC score among the Low LDL-C, Middle LDL-C and High LDL-C groups. CONCLUSIONS: We showed that the level of LDL-C was not associated with the presence or severity of CAD, which indicates that we need to screen by CCTA to prevent primary coronary events even if patients without anti-lipidemic therapies show low levels of LDL-C.

Regulation of DNA methylation using different tensions of double strands constructed in a defined DNA nanostructure.

A novel strategy for regulation of an enzymatic DNA modification reaction has been developed by employing a designed nanoscale DNA scaffold. DNA modification using enzymes often requires bending of specific DNA strands to facilitate the reaction. The DNA methylation enzyme EcoRI methyltransferase (M.EcoRI) bends double helix DNA by 55 degrees-59 degrees during the reaction with flipping out of the second adenine in the GAATTC sequence as the methyl transfer reaction proceeds. In this study, two different double helical tensions, tense and relaxed states of double helices, were created to control the methyl transfer reaction of M.EcoRI and examine the structural effect on the methylation. We designed and prepared a two-dimensional (2D) DNA scaffold named the "DNA frame" using the DNA origami method that accommodates two different lengths of the double-strand DNA fragments, a tense 64mer double strand and a relaxed 74mer double strand. Fast-scanning atomic force microscope (AFM) imaging revealed the different dynamic movement of the double-strand DNAs and complexes of M.EcoRI with 64mer and 74mer double-strand DNAs. After treatment of the double strands in the DNA frame with M.EcoRI and the subsequent digestion with restriction enzyme EcoRI (R.EcoRI), AFM analysis revealed that the 74mer double-strand DNA was not effectively cleaved compared with the 64mer double-strand DNA, indicating that the methylation preferentially occurred in the relaxed 74mer double-strand DNA compared with that in the tense 64mer double strand. Biochemical analysis of the methylation and specific digestion using a real-time PCR also supported the above results. These results indicate the importance of the structural flexibility for bending of the duplex DNA during the methyl transfer reaction with M.EcoRI. Therefore, the DNA methylation can be regulated using the structurally controlled double-strand DNAs constructed in the DNA frame nanostructure.

Visualization of dynamic conformational switching of the G-quadruplex in a DNA nanostructure.

We herein report the real-time observation of G-quadruplex formation by monitoring the G-quadruplex-induced global change of two duplexes incorporated in a DNA nanoscaffold. The introduced G-rich strands formed an interstrand (3 + 1) G-quadruplex structure in the presence of K(+), and the formed four-stranded structure was disrupted by removal of K(+). These conformational changes were visualized in a nanoscaffold in real-time with fast-scanning atomic force microscopy.

Characteristic effect of an anticancer dinuclear platinum(II) complex on the higher-order structure of DNA.

It is known that a 1,2,3-triazolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH(3))(2)}(2)(micro-OH)(micro-1,2,3-ta-N (1),N (2))](NO(3))(2) (AMTA), shows high in vitro cytotoxicity against several human tumor cell lines and circumvents cross-resistance to cisplatin. In the present study, we examined a dose- and time-dependent effect of AMTA on the higher-order structure of a large DNA, T4 phage DNA (166 kbp), by adapting single-molecule observation with fluorescence microscopy. It was found that AMTA induces the shrinking of DNA into a compact state with a much higher potency than cisplatin. From a quantitative analysis of the Brownian motion of individual DNA molecules in solution, it became clear that the density of a DNA segment in the compact state is about 2,000 times greater than that in the absence of AMTA. Circular dichroism spectra suggested that AMTA causes a transition from the B to the C form in the secondary structure of DNA, which is characterized by fast and slow processes. Electrophoretic measurements indicated that the binding of AMTA to supercoiled DNA induces unwinding of the double helix. Our results indicate that AMTA acts on DNA through both electrostatic interaction and coordination binding; the former causes a fast change in the secondary structure from the B to the C form, whereas the latter promotes shrinking in the higher-order structure as a relatively slow kinetic process. The shrinking effect of AMTA on DNA is attributable to the possible increase in the number of bridges along a DNA molecule. It is concluded that AMTA interacts with DNA in a manner markedly different from that of cisplatin.

Programmed-assembly system using DNA jigsaw pieces.

A novel method for assembling multiple DNA origami structures has been developed by using designed 2D DNA origami rectangles, so-called "DNA jigsaw pieces" that have sequence-programmed connectors. Shape and sequence complementarity were introduced to the concavity and convex connectors in the DNA rectangles for selective connection with the help of nonselective pi-stacking interactions between the side edges of the DNA jigsaw piece structures. Single DNA jigsaw piece units were assembled into unidirectional nanostructures with the correct alignment and uniform orientation. Three and five different DNA jigsaw pieces were assembled into predesigned and ordered nanostructures in a programmed fashion. Finally, three-, four-, and five-letter words have been displayed by using this programmed DNA jigsaw piece system.