DNA Hydrogels with Programmable Condensation, Expansion, and Degradation for Molecular Carriers

Molecular carriers are necessary for the controlled release of drugs and genes to achieve the desired therapeutic outcomes. DNA hydrogels can be a promising candidate in this application with their distinctive sequence-dependent programmability, which allows precise encapsulation of specific cargo molecules and stimuli-responsive release of them at the target. However, DNA hydrogels are inherently susceptible to the degradation of nucleases, making them vulnerable in a physiological environment. To be an effective molecular carrier, DNA hydrogels should be able to protect encapsulated cargo molecules until they reach the target and release them once they are reached. Here, we develop a simple way of controlling the enzyme resistance of DNA hydrogels for cargo protection and release by using cation-mediated condensation and expansion. We found that DNA hydrogels condensed by spermine are highly resistant to enzymatic degradation. They become degradable again if expanded back to their original, uncondensed state by sodium ions interfering with the interaction between spermine and DNA. These controllable condensation, expansion, and degradation of DNA hydrogels pave the way for the development of DNA hydrogels as an effective molecular carrier

Controlling Chiroptical Responses via Chemo-Mechanical Deformation of DNA Origami Structures

DNA origami-based templates have been widely used to fabricate chiral plasmonic metamaterials due to their precise control of the placement of nanoparticles (NPs) in a desired configuration. However, achieving various chiroptical responses inevitably requires a change in the structure of DNA origami-based templates or binding sites on them, leading to the use of significantly different sets of DNA strands. Here, we propose an approach to controlling various chiroptical responses with a single DNA origami design using its chemo-mechanical deformation induced by DNA intercalators. The chiroptical response could be finely tuned by altering the concentration of intercalators only. The silver (Ag) enhancement was used to amplify the chiroptical signal by enlarging NPs and to maintain it by stiffening the template DNA structure. Furthermore, the sensitivity in the chiroptical signal change to the concentration of intercalators could be modulated by the type of intercalator, the mixture of two intercalators, and the stiffness of DNA origami structures. This approach would be useful in a variety of optical applications that require programmed spatial modification of chiroptical responses

The oligomeric structure of the <i>Bh</i>ArgR and structural comparison with other homologous proteins.

<p>(A) The interface of the <i>Bh</i>ArgR trimer makes a hydrophobic core by the highly conserved residues Leu93, Leu129, and Ile122 of each subunit along the three-fold axis (left panel). The hydrophobic core is reinforced by the adjacent residues Val95, M119, and Ile131 of each subunit (right panel). (B) The hydrogen bond interactions are shown around the hydrophobic core in the <i>Bh</i>ArgR trimer. (C) Comparison with the N-terminal domains based on superposition of C-terminal domains of <i>Bh</i>ArgR (in green) and <i>Bst</i>ArgR (in blue). (D) Comparison with the trimeric structure of <i>Bh</i>ArgR and other homologous proteins based on superposition of C-terminal domains (<i>Bst</i>ArgR in blue; <i>Bsu</i>AhrC in violet).</p

Structural solution and Refinement.

<p>Structural solution and Refinement.</p

Structural Analysis and Insights into the Oligomeric State of an Arginine-Dependent Transcriptional Regulator from <i>Bacillus halodurans</i>

<div><p>The arginine repressor (ArgR) is an arginine-dependent transcription factor that regulates the expression of genes encoding proteins involved in the arginine biosynthesis and catabolic pathways. ArgR is a functional homolog of the arginine-dependent repressor/activator AhrC from <i>Bacillus subtilis</i>, and belongs to the ArgR/AhrC family of transcriptional regulators. In this research, we determined the structure of the ArgR (Bh2777) from <i>Bacillus halodurans</i> at 2.41 Å resolution by X-ray crystallography. The ArgR from <i>B</i>. <i>halodurans</i> appeared to be a trimer in a size exclusion column and in the crystal structure. However, it formed a hexamer in the presence of L-arginine in multi-angle light scattering (MALS) studies, indicating the oligomerization state was dependent on the presence of L-arginine. The trimeric structure showed that the C-terminal domains form the core, which was made by inter-subunit interactions mainly through hydrophobic contacts, while the N-terminal domains containing a winged helix-turn-helix DNA binding motif were arranged around the periphery. The arrangement of trimeric structure in the <i>B</i>. <i>halodurans</i> ArgR was different from those of other ArgR homologs previously reported. We finally showed that the <i>B</i>. <i>halodurans</i> ArgR has an arginine-dependent DNA binding property by an electrophoretic mobility shift assay.</p></div

The overall structure of <i>Bh</i>ArgR.

<p>(A) A multiple sequence alignment of <i>Bh</i>ArgR and represented homologous ArgR proteins from <i>B</i>. <i>stearothermophilus</i>, <i>B</i>. <i>subtilis</i>, <i>E</i>. <i>coli</i>, and <i>M</i>. <i>tuberculosis</i>. Every 10th residue is shown above the sequence of <i>Bh</i>ArgR. Highly conserved residues and partially conserved residues are shaded in black and grey, respectively. The residues involved in trimeric core with hydrophobic interactions and hydrogen bonds are indicated as black and red closed triangles, respectively. (B) The overall structure of <i>Bh</i>ArgR monomer. (C) The trimeric structure of <i>Bh</i>ArgR generated by a crystallographic three-fold symmetry through C-terminal domain (Each subunit is coloured in green, red, and yellow).</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Structural Analysis of Thymidylate Synthase from Kaposi’s Sarcoma-Associated Herpesvirus with the Anticancer Drug Raltitrexed

<div><p>Kaposi’s sarcoma-associated herpesvirus (KSHV) is a highly infectious human herpesvirus that causes Kaposi’s sarcoma. KSHV encodes functional thymidylate synthase, which is a target for anticancer drugs such as raltitrexed or 5-fluorouracil. Thymidylate synthase catalyzes the conversion of 2′-deoxyuridine-5′-monophosphate (dUMP) to thymidine-5′-monophosphate (dTMP) using 5,10-methylenetetrahydrofolate (mTHF) as a co-substrate. The crystal structures of thymidylate synthase from KSHV (apo), complexes with dUMP (binary), and complexes with both dUMP and raltitrexed (ternary) were determined at 1.7 Å, 2.0 Å, and 2.4 Å, respectively. While the ternary complex structures of human thymidylate synthase and <i>E</i>. <i>coli</i> thymidylate synthase had a closed conformation, the ternary complex structure of KSHV thymidylate synthase was observed in an open conformation, similar to that of rat thymidylate synthase. The complex structures of KSHV thymidylate synthase did not have a covalent bond between the sulfhydryl group of Cys219 and C6 atom of dUMP, unlike the human thymidylate synthase. The catalytic Cys residue demonstrated a dual conformation in the apo structure, and its sulfhydryl group was oriented toward the C6 atom of dUMP with no covalent bond upon ligand binding in the complex structures. These structural data provide the potential use of antifolates such as raltitrexed as a viral induced anticancer drug and structural basis to design drugs for targeting the thymidylate synthase of KSHV.</p></div

Facile Synthesis of Conductive Polypyrrole Wrinkle Topographies on Polydimethylsiloxane via a Swelling–Deswelling Process and Their Potential Uses in Tissue Engineering

Electrically conducting biomaterials have gained great attention in various biomedical studies especially to influence cell and tissue responses. In addition, wrinkling can present a unique topography that can modulate cell–material interactions. In this study, we developed a simple method to create wrinkle topographies of conductive polypyrrole (wPPy) on soft polydimethylsiloxane surfaces via a swelling–deswelling process during and after PPy polymerization and by varying the thickness of the PPy top layers. As a result, various features of wPPy in the range of the nano- and microscales were successfully obtained. In vitro cell culture studies with NIH 3T3 fibroblasts and PC12 neuronal cells indicated that the conductive wrinkle topographies promote cell adhesion and neurite outgrowth of PC12 cells. Our studies help to elucidate the design of the surface coating and patterning of conducting polymers, which will enable us to simultaneously provide topographical and electrical signals to improve cell–surface interactions for potential tissue-engineering applications

Ligand binding sites in kTS structures.

<p>(A), (B), and (C) represents apo (olive), binary complex (pink), and ternary complex kTS (cyan) structures, respectively. Atoms N, H, O of ligands are represented in blue, gray, and red, respectively. Atom C is colored in yellow in dUMP and chartreuse in raltitrexed. <i>2Fo-Fc</i> maps of catalytic Cys219, phosphate, dUMP, and raltitrexed are shown in apo, binary complex, and ternary complex kTS structures, respectively.</p