Direct Observation and Analysis of TET-mediated Oxidation Processes in a DNA Origami Nanochip

DNA methylation and demethylation play a key role in the epigenetic regulation of gene expression; however, a series of oxidation reactions of 5-methyl cytosine (5mC) mediated by ten-eleven translocation (TET) enzymes driving demethylation process are yet to be uncovered. To elucidate the relationship between the oxidative processes and structural factors of DNA, we analysed the behavior of TET-mediated 5mC-oxidation by incorporating structural stress onto a substrate double-stranded DNA (dsDNA) using a DNA origami nanochip. The reactions and behaviors of TET enzymes were systematically monitored by biochemical analysis and single-molecule observation using atomic force microscopy (AFM). A reformative frame-like DNA origami was established to allow the incorporation of dsDNAs as 5mC-containing substrates in parallel orientations. We tested the potential effect of dsDNAs present in the tense and relaxed states within a DNA nanochip on TET oxidation. Based on enzyme binding and the detection of oxidation reactions within the DNA nanochip, it was revealed that TET preferred a relaxed substrate regardless of the modification types of 5-oxidated-methyl cytosine. Strikingly, when a multi-5mCG sites model was deployed to further characterize substrate preferences of TET, TET preferred the fully methylated site over the hemi-methylated site. This analytical modality also permits the direct observations of dynamic movements of TET such as sliding and interstrand transfer by high-speed AFM. In addition, the thymine DNA glycosylase-mediated base excision repair process was characterized in the DNA nanochip. Thus, we have convincingly established the system's ability to physically regulate enzymatic reactions, which could prove useful for the observation and characterization of coordinated DNA demethylation processes at the nanoscale

DNA density-dependent uptake of DNA origami-based two-or three-dimensional nanostructures by immune cells

DNA nanostructures are expected to be applied for targeted drug delivery to immune cells. However, the structural properties of DNA nanostructures required for the delivery have not fully been elucidated. In this study, we focused on the DNA density that can be important for the their recognition and uptake by immune cells. To examine this, DNA nanostructures with almost identical molecular weights and structural flexibility, but with different shapes and DNA densities, were designed using DNA origami technology. We compared the following five types of DNA nanostructures, all of which consisted of ten DNA helices using an identical circular, single-stranded scaffold and staples. Rec180 had a rectangular-shaped, almost flat structure. Rec90, Rec50 and Rec0 were bent forms of Rec180 at the center by 90, 50 or 0 degrees, respectively. Rec50/50 has two bends of 50 degrees each so that the both ends stick together to form a triangular prism shape. The fluctuation, or flexibility, of these DNA nanostructures under solution conditions was estimated using CanDo software. The DNA density estimated from the average distance between any two of the ten DNA helices in the DNA nanostructures was different among them; Rec50, Rec0 and Rec50/50 had a higher density than Rec180 and Rec90. Agarose gel electrophoresis and atomic force microscopy showed that all of the nanostructures were prepared with high yield. Flow cytometry analysis revealed that the uptake of DNA nanostructures by murine macrophage-like RAW264.7 cells was higher for those with higher DNA density than those with low density. There was a positive correlation between the density and cellular uptake. These results indicate that DNA nanostructures with high DNA density are suitable for delivery to immune cells

Single-molecule observations of RNA-RNA kissing interactions in a DNA nanostructure

RNA molecules uniquely form a complex through specific hairpin loops, called a kissing complex. The kissing complex is widely investigated and used for the construction of RNA nanostructures. Molecular switches have also been created by combining a kissing loop and a ligand-binding aptamer to control the interactions of RNA molecules. In this study, we incorporated two kinds of RNA molecules into a DNA origami structure and used atomic force microscopy to observe their ligand-responsive interactions at the single-molecule level. We used a designed RNA aptamer called GTPswitch, which has a guanosine triphosphate (GTP) responsive domain and can bind to the target RNA hairpin named Aptakiss in the presence of GTP. We observed shape changes of the DNA/RNA strands in the DNA origami, which are induced by the GTPswitch, into two different shapes in the absence and presence of GTP, respectively. We also found that the switching function in the nanospace could be improved by using a cover strand over the kissing loop of the GTPswitch or by deleting one base from this kissing loop. These newly designed ligand-responsive aptamers can be used for the controlled assembly of the various DNA and RNA nanostructures

Crossovers within a short DNA sequence indicate a long evolutionary history of the APRT*J mutation

Adenine phosphoribosyltransferase (APRT) deficiency causing 2,8-dihydroxyadenine urolithiasis and renal failure is present at a high frequency among the Japanese but not other ethnic groups. A special type of mutant allele, designated APRT*J , with a nucleotide substitution at codon 136 from ATG (Met) to ACG (Thr) is carried by approximately 79% of all Japanese 2,8-dihydroxyadenine urolithiasis patients. We analyzed mutant alleles of 39 APRT deficient patients using a specific oligonucleotide hybridization method after in vitro amplification of a part of the genomic APRT sequence. We found that 24 had only APRT*J alleles. Determination of the haplotypes of 194 APRT alleles from control Japanese subjects and of the 48 different APRT*J alleles indicated that normal alleles occur in four major haplotypes, whereas all APRT*J alleles occur in only two. These results suggest that all APRT*J alleles have a single origin and that this mutant sequence has been maintained for a long period, as calculated from the frequency of the recombinant alleles.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47628/1/439_2004_Article_BF00193582.pd

Enhanced boiling surface with hydrophobic circle spots evaporator of looped thermosiphon

Heat transfer characteristic of a closed two-phase thermosiphon with enhanced boiling surface is studied and compared with that of a copper mirror surface. Two-phase cooling improves heat transfer coefficient (HTC) a lot compared to singlephase liquid cooling. The evaporator surfaces coated with a pattern of hydrophobic circle spots (non-electroplating, 0.5 2 mm in diameter and 1.5 3 mm in pitch) achieve very high heat transfer coefficient and lower the incipience temperature overshoot using water as the working fluid. Sub-atmospheric boiling on the hydrophobic spot-coated surface shows a much better heat transfer performance. Tests with heat loads (30 W to 260 W) revealed the optimum thermosiphon performance. Hydrophobic circle spots coated surface with diameter 1 mm, pitch 1.5 mm achieves the maximum heat transfer enhancement with the boiling thermal resistance as low as 0.03 K/W.Papers presented at the 13th International Conference on Heat Transfer, Fluid Mechanics and Thermodynamics, Portoroz, Slovenia on 17-19 July 2017 .International centre for heat and mass transfer.American society of thermal and fluids engineers

The nuclear interaction at Oklo 2 billion years ago

We re-examine the effort to constrain the time-variability of the coupling constants of the fundamental interactions by studying the anomalous isotopic abundance of Sm observed at the remnants of the natural reactors which were in operation at Oklo about 2 billion years ago, in terms of a possible deviation of the resonance energy from the value observed today. We rely on new samples that were carefully collected to minimize natural contamination and also on a careful temperature estimate of the reactors. We obtain the upper bound $(-0.2\pm 0.8)\times 10^{-17}$ ${\rm y}^{-1}$ on the fractional rate of change of the electromagnetic as well as the strong interaction coupling constants. Our result basically agrees with and even suggests some improvement of the result due recently to Damour and Dyson. Strictly speaking, however, we find another range of the resonance energy shift indicating a nonzero time variation of the constants. We find a rather strong but still tentative indication that this range can be ruled out by including the Gd data, for which it is essential to take the effect of contamination into account.Comment: 20 pages LaTex including 6 figures. Theoretical interpretation changed. More detailed discussions on the temperature estimate also adde