Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases

Dimethyl fumarate (DMF) has been applied for decades in the treatment of psoriasis and now also multiple sclerosis. However, the mechanism of action has remained obscure and involves high dose over long time of this small, reactive compound implicating many potential targets. Based on a 1.9 Å resolution crystal structure of the C-terminal kinase domain of the mouse p90 Ribosomal S6 Kinase 2 (RSK2) inhibited by DMF we describe a central binding site in RSKs and the closely related Mitogen and Stress-activated Kinases (MSKs). DMF reacts covalently as a Michael acceptor to a conserved cysteine residue in the αF-helix of RSK/MSKs. Binding of DMF prevents the activation loop of the kinase from engaging substrate, and stabilizes an auto-inhibitory αL-helix, thus pointing to an effective, allosteric mechanism of kinase inhibition. The biochemical and cell biological characteristics of DMF inhibition of RSK/MSKs are consistent with the clinical protocols of DMF treatment.</p

Phosphines as Efficient Dioxygen Scavengers in Nitrous Oxide Sensors

A current challenge for development of amperometric sensors for the greenhouse gas nitrous oxide (N₂O) is their sensitivity toward dioxygen and trace water. The need for aqueous dioxygen scavengers in front of the sensor implies a background signal from penetrating water vapor. In this paper, we introduce substituted phosphines as dioxygen scavengers and demonstrate the application in a dioxygen-insensitive N₂O sensor. Suitably substituted phosphines have been synthesized to achieve good solubility properties in the electrochemically inert solvent propylene carbonate. Several sensors with and without physical separation of the sensing and dioxygen scavenging compartments were made and compared to current commercial sensors. The use of phosphines soluble in organic solvents as dioxygen scavengers yielded a higher sensitivity, albeit with longer response time. Proof-of-concept N₂O sensors without the physically separated dioxygen scavenger chamber showed a greatly enhanced sensitivity with a comparable response time, thus demonstrating the possibility for greatly simplified sensor construction

Folding double-stranded DNA into designed shapes with triplex-forming oligonucleotides

The folding of double-stranded DNA around histones is a central mechanism in eukaryotic cells for compacting the genetic information into chromosomes. Very few artificial methods are available for controlling the shape of dsDNA at any level, whereas several artificial methods have been developed to efficiently organize single-stranded DNA and RNA into a variety of well-defined nanostructures by programmed self-assembly. Here, we show how long double-stranded DNA sequences can be spatially organized by triplex-forming oligonucleotides (TFOs), which bridge two or more encoded polypurine domains. The linearized or plasmid dsDNA is compacted into antiparallel folds, which enables the formation of raster-filled 2D shapes and 3D structures with either square or hexagonal organizations. Contrary to ssDNA, dsDNA has inherent rigidity which alleviates the requirement to saturate a structure with TFO strands, yet the TFOs are still able to bend the dsDNA controllably and steeply up to 180° over 6 bp. The majority of structures investigated here are formed by Hoogsteen interactions which require pH = 5-6, however, the methodology is also applied with reverse Hoogsteen interactions at physiological pH. In both cases, the DNA triplexes render pure polypurine scaffolded structures resistant to DNase I

Programmed Switching of Single Polymer Conformation on DNA Origami

DNA nanotechnology offers precise geometrical control of the positioning of materials, and it is increasingly also being used in the development of nanomechanical devices. Here we describe the development of a nanomechanical device that allows switching of the position of a single-molecule conjugated polymer. The polymer is functionalized with short single-stranded (ss) DNA strands that extend from the backbone of the polymer and serve as handles. The DNA polymer conjugate can be aligned on DNA origami in three well-defined geometries (straight line, left-turned, and right-turned pattern) by DNA hybridization directed by single-stranded guiding strands and ssDNA tracks extending from the origami surface and polymer handle. We demonstrate switching of a conjugated organic polymer conformation between left- and right-turned conformations of the polymer on DNA origami based on toehold-mediated strand displacement. The switching is observed by atomic force microscopy and by Förster resonance energy transfer between the polymer and two different organic dyes positioned in close proximity to the respective patterns. Using this method, the polymer conformation can be switched six times successively. This controlled nanomechanical switching of conjugated organic polymer conformation demonstrates unique control of the shape of a single polymer molecule, and it may constitute a new component for the development of reconfigurable nanophotonic and nanoelectronic devices

Synthesis of Dopamine and Serotonin Derivatives for Immobilization on a Solid Support

The two important neurotransmitters dopamine and serotonin are synthesized with short PEG tethers and immobilized on a magnetic solid support. The tether is attached to the aromatic moiety of the neurotransmitters to conserve their original functional groups. This approach causes minimal alteration of the original structure with the aim of optimizing the immobilized neurotransmitters for aptamer selection by SELEX. For the dopamine derivative, the tether is attached to the aromatic core of a dopamine precursor by the Sonogashira reaction. For serotonin, a link to the indole core is introduced by a Claisen rearrangement from the allylated phenol moiety of serotonin. The tethers are azide-functionalized, which enables coupling to alkyne-modified magnetic beads. The coupling to the magnetic beads is quantified by UV spectroscopy using Fmoc-monitoring of the immobilized dopamine and serotonin derivatives

A Yoctoliter-Scale DNA Reactor for Small-Molecule Evolution

The center of DNA three-way junctions, constituting a yoctoliter (10⁻²⁴ L) volume, is applied as an efficient reactor to create DNA-encoded libraries of chemical products. Amino acids and short peptides are linked to oligonucleotides via cleavable and noncleavable linkers. The oligonucleotide sequences contain two universal assembling domains at the center and a distal codon sequence specific for the attached building block. Stepwise self-assembly and chemical reactions of these conjugates in a combinatorial fashion create a library of pentapeptides in DNA three-way junctions in a single reaction vessel. We demonstrate the formation of an evenly distributed library of 100 peptides. Each library member contains a short synthetic peptide attached to a unique genetic code creating the necessary “genotype−phenotype” linkage essential to the process of <i>in vitro</i> molecular evolution. Selective enrichment of the [Leu]-enkephalin peptide from an original frequency of 1 in 10 million in a model library to a final frequency of 1.7% in only two rounds of affinity selection is described and demonstrates successful molecular evolution for a non-natural system

Direct Visualization of Transient Thermal Response of a DNA Origami

The DNA origami approach enables the construction of complex objects from DNA strands. A fundamental understanding of the kinetics and thermodynamics of DNA origami assembly is extremely important for building large DNA structures with multifunctionality. Here both experimental and theoretical studies of DNA origami melting were carried out in order to reveal the reversible association/disassociation process. Furthermore, by careful control of the temperature cycling via in situ thermally controlled atomic force microscopy, the self-assembly process of a rectangular DNA origami tile was directly visualized, unveiling key mechanisms underlying their structural and thermodynamic features