Recently, it has been confirmed that long duplex DNA molecules with sizes larger than several tens of kilo-base pairs (kbp), exhibit a discrete conformational transition from an elongated coil state to a compact globule state upon the addition of various kinds of chemical species that usually induce DNA condensation. In this study, we performed a single-molecule observation on a large DNA, Lambda ZAP II DNA (ca. 41 kbp), in a solution containing RNA polymerase and substrates along with spermine, a tetravalent cation, at different concentrations, by use of fluorescence staining of both DNA and RNA. We found that transcription, or RNA production, is completely inhibited in the compact state, but is actively performed in the unfolded coil state. Such an all-or-none effect on transcriptional activity induced by the discrete conformational transition of single DNA molecules is discussed in relation to the mechanism of the regulation of large-scale genetic activity.
The front cover picture shows selective localization of F-actin (polymerized filament) and double-stranded DNA within a cell-sized aqueous/aqueous droplet that was generated spontaneously through simple mixing with binary hydrophilic polymers, poly(ethylene glycol) and dextran (DEX). Interestingly, the manner of localization of these biomacromolecules switches depending on their molecular size. Long double-stranded DNA molecules, above the size of several tens of kilo base pairs, are located solely inside DEX-rich droplets. In contrast, short oligomeric single-stranded DNA exists homogeneously without any apparent concentration difference. For the actins, G-actins (monomeric) are evenly distributed, but F-actins are localized inside DEX-rich droplets. As in the picture, the existence of both F-actin and DNA creates a specific structure in which DNA molecules are depleted by actin fibers arranged in parallel, something like the morphology of cell mitosis. The self-emergence of characteristic morphologies in cell-sized droplets could shed light on the underlying mechanism of the spatiotemporal self-organization of living cellular systems. More information can be found in the communication by K. Takiguchi, K. Tsumoto, K. Yoshikawa, et al. on page 1370 in Issue 13, 2018 (DOI: 10.1002/cbic.201800066).
Abstract DNA is an excellent material for constructing self‐assembled nano/microstructures. Owing to the widespread use of DNA as a building block in laboratories and industry, it is desirable to increase the efficiency of all steps involved in producing self‐assembled DNA structures. One of the bottlenecks is the purification required to separate the excess components from the target structures. This paper describes a purification method based on the fractionation by water‐in‐water (W/W) droplets composed of phase‐separated dextran‐rich droplets in a polyethylene glycol (PEG)‐rich continuous phase. The dextran‐rich droplets facilitate the selective uptake of self‐assembled DNA nano/microstructures and allow the separation of the target structure. This study investigates the ability to purify DNA origami, DNA hydrogels, and DNA microtubes. The W/W‐droplet fractionation allows the purification of structures of a broad size spectrum without changes to the protocol. By quantifying the activity of deoxyribozyme‐modified DNA origami after W/W‐droplet purification, this study demonstrates that this method sufficiently preserves the accessibility to the surface of a functional DNA nanostructure. It is considered that the W/W‐droplet fractionation could become one of the standard methods for the purification of self‐assembled DNA nano/microstructures for biomedical and nanotechnology applications owing to its low cost and simplicity.
Three foldases—the apical domain of GroEL (mini‐chaperone) and two oxidoreductases (DsbA and DsbC) from Escherichia coli—were studied in refolding a protein with immunoglobulin fold (immunoglobulin‐folded protein) that had been produced as inclusion bodies in E.coli. The foldases promoted the refolding of single‐chain antibody fragments from denaturant‐solubilized and reduced inclusion bodies in vitro, and also effectively functioned as alternatives for labilizing agent and oxidizing reagent in the stepwise dialysis system. Immobilization of the oxidoreductases enhanced refolding and recovery of functional single‐chain antibody in the dialysis system, suggesting that immobilized oxidoreductases can be used as an effective additive for refolding immunoglobulin‐folded proteins in vitro.
