Structural Studies of a Complex of a CAG/CTG Repeat Sequence-Specific Binding Molecule and A-A-Mismatch-Containing DNA.

Triplet repeat diseases are caused by the abnormal elongation of repeated sequences comprising three bases. In particular, the elongation of CAG/CTG repeat sequences is thought to result in conditions such as Huntington's disease and myotonic dystrophy type 1. Although the causes of these diseases are known, fundamental treatments have not been established, and specific drugs are expected to be developed.

Multivalent dendritic DNA aptamer molecules for the enhancement of therapeutic effects.

Dendritic DNA molecules, referred to as DNA dendrons, consist of multiple covalently linked strands and are expected to improve the cellular uptake and potency of therapeutic oligonucleotides because of their multivalency. In this study, we developed an efficient synthetic method for producing DNA dendrons using strain-promoted azide-alkyne cycloaddition. Integration of the antitumor aptamer AS1411 into DNA dendrons enhanced cellular uptake and antiproliferative activity in cancer cells.

Structural Expansion of Catalytic RNA Nanostructures through Oligomerization of a Cyclic Trimer of Engineered Ribozymes.

The multimolecular assembly of three-dimensionally structured proteins forms their quaternary structures, some of which have high geometric symmetry. The size and complexity of protein quaternary structures often increase in a hierarchical manner, with simpler, smaller structures serving as units for larger quaternary structures. In this study, we exploited oligomerization of a ribozyme cyclic trimer to achieve larger ribozyme-based RNA assembly.

Photocontrolled DNA nanotubes as stiffness tunable matrices for controlling cellular behavior.

Cell behavior is determined by a variety of properties of the extracellular environment like ligand spacing, nanotopography, and matrix stiffness. Matrix stiffness changes occur during many biological processes like wound healing, tumorigenesis, and development. These spatio-temporal dynamic changes in stiffness can cause significant changes in cell morphology, cell signaling, migration, cytoskeleton .

Catalytic RNA Oligomers Formed by Co-Oligomerization of a Pair of Bimolecular RNase P Ribozymes.

Naturally occurring ribozymes with a modular architecture are promising platforms for construction of RNA nanostructures because modular redesign enables their oligomerization. The resulting RNA nanostructures can exhibit the catalytic function of the parent ribozyme in an assembly dependent manner. In this study, we designed and constructed open-form oligomers of a bimolecular form of an RNase P ribozyme.

Box-shaped ribozyme octamer formed by face-to-face dimerization of a pair of square-shaped ribozyme tetramers.

Naturally occurring ribozymes with defined three-dimensional (3D) structures serve as promising platforms for the design and construction of artificial RNA nanostructures. We constructed a hexameric ribozyme nanostructure by face-to-face dimerization of a pair of triangular ribozyme trimers, unit RNAs of which were derived from the Tetrahymena group I ribozyme. In this study, we have expanded the dimerization strategy to a square-shaped ribozyme tetramer by introducing four pillar units.

A Hexameric Ribozyme Nanostructure Formed by Double-Decker Assembly of a Pair of Triangular Ribozyme Trimers.

The modular architecture of naturally occurring ribozymes makes them a promising class of structural platform for the design and assembly of three-dimensional (3D) RNA nanostructures, into which the catalytic ability of the platform ribozyme can be installed. We have constructed and analyzed RNA nanostructures with polygonal-shaped (closed) ribozyme oligomers by assembling unit RNAs derived from the Tetrahymena group I intron with a typical modular architecture. In this study, we dimerized ribozyme trimers with a triangular shape by introducing three pillar units.

Non-invasive Regulation of Cellular Morphology Using a Photoswitchable Mechanical DNA Polymer.

The extracellular matrix (ECM) in which the cells reside provides a dynamic and reversible environment. Spatiotemporal cues are essential when cells are undergoing morphogenesis, repair and differentiation. Emulation of such an intricate system with reversible presentation of nanoscale cues can help us better understand cellular processes and can allow the precise manipulation of cell function in vitro.

HBD1 protein with a tandem repeat of two HMG-box domains is a DNA clip to organize chloroplast nucleoids in .

Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA-protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p).

Flexible Assembly of Engineered Tetrahymena Ribozymes Forming Polygonal RNA Nanostructures with Catalytic Ability.

Ribozymes with modular architecture constitute an attractive class of structural platforms for design and construction of nucleic acid nanostructures with biological functions. Through modular engineering of the Tetrahymena ribozyme, we have designed unit RNAs (L-RNAs), assembly of which formed ribozyme-based closed trimers and closed tetramers. Their catalytic activity was dependent on oligomer formation.

Photocontrolled DNA Origami Assembly by Using Two Photoswitches.

Stimuli-responsive switching molecules have been widely investigated for the purpose of the mechanical control of biomolecules. Recently developed arylazopyrazole (AAP) shows photoisomerization activity, displaying a faster response to light-induced conformational changes and unique absorption spectral properties compared with those of conventionally used azobenzene. Herein, it is demonstrated that AAP can be used as a photoswitching molecule to control photoinduced assembly and disassembly of DNA origami nanostructures.

Direct Observation of Dynamic Interactions between Orientation-Controlled Nucleosomes in a DNA Origami Frame.

The nucleosome is one of the most fundamental units involved in gene expression and consequent cell development, differentiation, and expression of cell functions. We report here a method to place reconstituted nucleosomes into a DNA origami frame for direct observation using high-speed atomic-force microscopy (HS-AFM). By using this method, multiple nucleosomes can be incorporated into a DNA origami frame and real-time movement of nucleosomes can be visualized.

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.

Catalytic RNA nano-objects formed by self-assembly of group I ribozyme dimers serving as unit structures.

Ribozymes with modular structures are attractive platforms for the construction of nanoscale RNA objects with biological functions. We designed group I ribozyme dimers as unit ribozyme dimers (Urds), which self-assembled to form their polymeric states and also oligomeric states with defined numbers of Urds. Assembly of Urds yielded catalytic ability of a pair of distinct ribozyme units to cleave two distinct substrates.

Folding of single-stranded circular DNA into rigid rectangular DNA accelerates its cellular uptake.

Despite the importance of the interaction between DNA and cells for its biological activity, little is known about exactly how DNA interacts with cells. To elucidate the relationship between the structural properties of DNA and its cellular uptake, a single-stranded circular DNA of 1801 bases was designed and folded into a series of rectangular DNA (RecDNA) nanostructures with different rigidities using DNA origami technology. Interactions between these structures and cells were evaluated using mouse macrophage-like RAW264.

Effects of Physical Damage in the Intermediate Phase on the Progression of Amyloid β Fibrillization.

Understanding the mechanism responsible for the progression of amyloid deposition is important for developing methods to suppress this process in the treatment of Alzheimer's disease. The effects of physical damage during the transition phase of amyloid β fibril formation are unclear. In this study, we used high-speed atomic force microscopy to investigate the effects of damage to the intermediates of amyloid β in real time.

Translation-dependent unwinding of stem-loops by UPF1 licenses Regnase-1 to degrade inflammatory mRNAs.

Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem-loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem-loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem-loops, thus licensing Regnase-1 to proceed with RNA degradation.

Direct Observation and Analysis of the Dynamics of the Photoresponsive Transcription Factor GAL4.

Herein, the direct visualization of the dynamic interaction between a photoresponsive transcription factor fusion, GAL4-VVD, and DNA using high-speed atomic force microscopy (HS-AFM) is reported. A series of different GAL4-VVD movements, such as binding, sliding, stalling, and dissociation, was observed. Inter-strand jumping on two double-stranded (ds) DNAs was also observed.

A Photocaged DNA Nanocapsule for Controlled Unlocking and Opening inside the Cell.

We report a nanosized DNA capsule with a photoinducible mechanical unlocking system for creation of a carrier for delivery system to the cells. A photocage system was introduced into the nanocapsule (NC) for control of opening of the NC with photoirradiation. The opening of the NC was observed by atomic force microscopy (AFM), and the dynamic opening of the NC was examined by fluorescence recovery from the quenching.

Programming Rotary Motions with a Hexagonal DNA Nanomachine.

Biological macromolecular machines perform impressive mechanical movements. F-adenosine triphosphate (ATP) synthase uses a proton gradient to generate ATP through mechanical rotations. Here, a programmed hexagonal DNA nanomachine, in which a three-armed DNA nanostructure (TAN) can perform stepwise rotations in the confined nanospace powered by DNA fuels, is demonstrated.

Direct Observation of the Double-Stranded DNA Formation through Metal Ion-Mediated Base Pairing in the Nanoscale Structure.

This work demonstrates single-molecule imaging of metal-ion induced double-stranded DNA formation in DNA nanostructures. The formation of the metal ion-mediated base pairing in a DNA origami frame was examined using C-Ag-C and T-Hg-T metallo-base pairs. The target DNA strands containing consecutive C or T were incorporated into the DNA frame, and the binding was controlled by the addition of metal ions.

Decreased water activity in nanoconfinement contributes to the folding of G-quadruplex and i-motif structures.

Due to the small size of a nanoconfinement, the property of water contained inside is rather challenging to probe. Herein, we measured the amount of water molecules released during the folding of individual G-quadruplex and i-motif structures, from which water activities are estimated in the DNA nanocages prepared by 5 × 5 to 7 × 7 helix bundles (cross-sections, 9 × 9 to 15 × 15 nm). We found water activities decrease with reducing cage size.

DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K Channels.

In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes.