Quality Control of mRNA Vaccines by Synthetic Ribonucleases: Analysis of the Poly-A-Tail.

The effectivity and safety of mRNA vaccines critically depends on the presence of correct 5' caps and poly-A tails. Due to the high molecular mass of full-size mRNAs, however, the direct analysis by mass spectrometry is hardly possible. Here we describe the use of synthetic ribonucleases to cleave off 5' and 3' terminal fragments which can be further analyzed by HPLC or by LC-MS. Compared to existing methods (e.g. RNase H), the new approach uses robust catalysts, is free of sequence limitations, avoids metal ions and combines fast sample preparation with high precision of the cut.

Hybrid Organosulfur Network/MWCNT Composite Cathodes for Li-S Batteries.

Lithium-sulfur (Li-S) batteries hold a promising position as candidates for next-generation high-energy storage systems. Here, we combine inverse vulcanization of sulfur with multiwalled carbon nanotubes (MWCNTs) to increase the conductivity of cathode materials for Li-S batteries. The mixing process of inversely vulcanized sulfur copolymer networks with MWCNTs is aided by shear in a two-roll mill to take advantage of the soft nature of the copolymer. The high-throughput mixing method demands a source of conductive carbon that can be intimately mixed with the S copolymer, rendering MWCNTs an excellent choice for this purpose. The resulting sulfur copolymer network-MWCNTs composites were thoroughly characterized in terms of structure, chemical composition, thermal, and electronic transport properties, and finally evaluated by electrochemical benchmarking. These promising hybrids yielded electrodes with high sulfur content and demonstrate stable electrochemical performance exhibiting a specific capacity of ca. 550 mAh·gsulfur-1 (380 mAh·gelectrode-1) even after 500 charge-discharge cycles at specific current of 167 mA·g-1 (corresponds to 0.1C discharge rate), and thus are superior to melt-infiltrated reference samples.

Spin-Labeled Riboswitch Synthesized from a Protected TPA Phosphoramidite Building Block.

The nitroxide TPA (2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene) is an excellent spin label for EPR studies of RNA. Previous synthetic methods, however, are complicated and require special equipment. Herein, we describe a uridine derived phosphoramidite with a photocaged TPA unit attached. The light sensitive 2-nitrobenzyloxymethyl group can be removed in high yield by short irradiation at 365 nm. Based on this approach, a doubly spin-labeled 27mer neomycin sensing riboswitch was synthesized and studied by PELDOR. The overall thermal stability of the fold is not much reduced by TPA. In-line probing nevertheless detected changes in local mobility.

Exploring the Druggability of Conserved RNA Regulatory Elements in the SARS-CoV-2 Genome.

SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.

Vertical organic permeable dual-base transistors for logic circuits.

The main advantage of organic transistors with dual gates/bases is that the threshold voltages can be set as a function of the applied second gate/base bias, which is crucial for the application in logic gates and integrated circuits. However, incorporating a dual gate/base structure into an ultra-short channel vertical architecture represents a substantial challenge. Here, we realize a device concept of vertical organic permeable dual-base transistors, where the dual base electrodes can be used to tune the threshold voltages and change the on-currents. The detailed operation mechanisms are investigated by calibrated TCAD simulations. Finally, power-efficient logic circuits, e.g. inverter, NAND/AND computation functions are demonstrated with one single device operating at supply voltages of <2.0 V. We believe that this work offers a compact and technologically simple hardware platform with excellent application potential for vertical-channel organic transistors in complex logic circuits.

Dry-Jet Wet Spinning of Thermally Stable Lignin-Textile Grade Polyacrylonitrile Fibers Regenerated from Chloride-Based Ionic Liquids Compounds.

In this paper, we report on the use of amorphous lignin, a waste by-product of the paper industry, for the production of high performance carbon fibers (CF) as precursor with improved thermal stability and thermo-mechanical properties. The precursor was prepared by blending of lignin with polyacrylonitrile (PAN), which was previously dissolved in an ionic liquid. The fibers thus produced offered very high thermal stability as compared with the fiber consisting of pure PAN. The molecular compatibility, miscibility, and thermal stability of the system were studied by means of shear rheological measurements. The achieved mechanical properties were found to be related to the temperature-dependent relaxation time (consistence parameter) of the spinning dope and the diffusion kinetics of the ionic liquids from the fibers into the coagulation bath. Furthermore, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and dynamic mechanical tests (DMA) were utilized to understand in-depth the thermal and the stabilization kinetics of the developed fibers and the impact of lignin on the stabilization process of the fibers. Low molecular weight lignin increased the thermally induced physical shrinkage, suggesting disturbing effects on the semi-crystalline domains of the PAN matrix, and suppressed the chemically induced shrinkage of the fibers. The knowledge gained throughout the present paper allows summarizing a novel avenue to develop lignin-based CF designed with adjusted thermal stability.

Nanoparticle assembly under block copolymer confinement: The effect of nanoparticle size and confinement strength.

We investigate the self-assembly of cylinder-forming polystyrene-block-poly(4-vinylpyridine) (PS-b-P4VP) block copolymers (BCP) mixed with metal nanoparticles (NP) coated with short-chain polystyrene (PS) ligands. The NP formed hierarchical superstructures under confinement of cylindrical PS domains of PS-b-P4VP BCP. The complexity of NP superstructures was found to depend on the ratio between PS cylindrical domain size and NP size (DC/DNP). As the DC/DNP ratio increased, the number of NP layers normal to the cylinder axis also increased. However, the packing density of the NP decreased at higher DC/DNP. Furthermore, the morphology of the structures obtained during different solvent casting conditions revealed that the initial clustering of NP and micellization around these clusters act as a precursor for the subsequent formation of closely packed structures of NP in cylinders. The experimental results were further supported by modeling results obtained from molecular dynamics (MD) simulation. Based on MD simulations, we constructed structural phase diagram of nanoparticle assemblies in the presence of asymmetric diblock copolymers comprising short NP-attractive blocks. The MD simulation results indicate that NP undergo transition from spherical to cylindrical assemblies depending on the NP size, the overall concentration of components and the degree of affinity of the minor block to NP.

Redirection of miRNA-Argonaute Complexes to Specific Target Sites by Synthetic Adaptor Molecules.

Dysregulation of miRNAs is connected with a multitude of diseases for which antagomirs and miRNA replacement are discussed as therapeutic options. Here, we suggest an alternative concept based on the redirection of RISCs to non-native target sites. Metabolically stable DNA-LNA mixmers are used to mediate the binding of RISCs to mRNAs without any direct base complementarity to the presented guide RNA strand. Physical redirection of a dye-labeled miRNA model and of specific miRNA-programmed RISC fractions present in HeLa extracts is demonstrated by pull-down experiments with biotinylated capture oligonucleotides.

A Trisbenzimidazole Phosphoramidite Building Block Enables High-Yielding Syntheses of RNA-Cleaving Oligonucleotide Conjugates.

The RNA cleaving catalyst tris(2-aminobenzimidazole) when attached to the 5' terminus of oligonucleotides cuts complementary RNA strands in a highly site-specific manner. Conjugation was previously achieved by the acylation of an amino linker by an active ester of the catalyst. However, this procedure was low yielding and not reliable. Here, a phosphoramidite building block is described that can be coupled to oligonucleotides by manual solid phase synthesis in total yields around 85%. Based on this chemistry, we have now studied the impact of LNA (locked nucleic acids) nucleotides on the rates and the site-specificities of RNA cleaving conjugates. The highest reaction rates and the most precise cuts can be expected when the catalyst is attached to a strong 5' closing base pair and when the oligonucleotide contains several LNA units that are equally distributed in the strand. However, when placed in the 5' position, LNA building blocks tend to diminish the specificity of RNA cleavage.

Peptide Nucleic Acid Conjugates of Quinone Methide Precursors Alkylate Ribonucleic Acid after Activation with Light.

Quinone methide precursors 2 and 3 were protected with a photoreactive 2-nitrobenzyl group and conjugated to peptide nucleic acids (PNA) using a Huisgen click reaction. After brief irradiation at 365 nm, cross-linking with complementary RNA strands started and was analyzed with an ALFexpress sequencer. When this method was used, the gel temperature had a major influence on apparent rates. Quinone methides are known to form transient as well as stable bonds with nucleotides. Although both were detected at 25 °C, analysis at 57 °C only recorded the stable types of cross-links, suggesting much slower alkylation kinetics. Linker 11 allowed us to attach quinone methides to internal positions of the PNA/RNA duplex and to capture a model of miR-20a with good efficiency.

Genetic Code Expansion Facilitates Position-Selective Labeling of RNA for Biophysical Studies.

Nature relies on reading and synthesizing the genetic code with high fidelity. Nucleic acid building blocks that are orthogonal to the canonical A-T and G-C base-pairs are therefore uniquely suitable to facilitate position-specific labeling of nucleic acids. Here, we employ the orthogonal kappa-xanthosine-base-pair for in vitro transcription of labeled RNA. We devised an improved synthetic route to obtain the phosphoramidite of the deoxy-version of the kappa nucleoside in solid phase synthesis. From this DNA template, we demonstrate the reliable incorporation of xanthosine during in vitro transcription. Using NMR spectroscopy, we show that xanthosine introduces only minor structural changes in an RNA helix. We furthermore synthesized a clickable 7-deaza-xanthosine, which allows to site-specifically modify transcribed RNA molecules with fluorophores or other labels.

Site-Specific Cleavage of RNAs Derived from the PIM1 3'-UTR by a Metal-Free Artificial Ribonuclease.

Oligonucleotide conjugates of tris(2-aminobenzimidazole) have been reported previously to cleave complementary RNA strands with high levels of sequence and site specificity. The RNA substrates used in these studies were oligonucleotides not longer than 29-mers. Here we show that ~150⁻400-mer model transcripts derived from the 3'-untranslated region of the PIM1 mRNA reacted with rates and specificities comparable to those of short oligonucleotide substrates. The replacement of DNA by DNA/LNA mixmers further increased the cleavage rate. Tris(2-aminobenzimidazoles) were designed to interact with phosphates and phosphate esters. A cell, however, contains large amounts of phosphorylated species that may cause competitive inhibition of RNA cleavage. It is thus important to note that no loss in reaction rates was observed in phosphate buffer. This opens the way to in-cell applications for this type of artificial nuclease. Furthermore, we disclose a new synthetic method giving access to tris(2-aminobenzimidazoles) in multigram amounts.

Phosphoramidite building blocks with protected nitroxides for the synthesis of spin-labeled DNA and RNA.

TEMPO spin labels protected with 2-nitrobenzyloxymethyl groups were attached to the amino residues of three different nucleosides: deoxycytidine, deoxyadenosine, and adenosine. The corresponding phosphoramidites could be incorporated by unmodified standard procedures into four different self-complementary DNA and two RNA oligonucleotides. After photochemical removal of the protective group, elimination of formic aldehyde and spontaneous air oxidation, the nitroxide radicals were regenerated in high yield. The resulting spin-labeled palindromic duplexes could be directly investigated by PELDOR spectroscopy without further purification steps. Spin-spin distances measured by PELDOR correspond well to the values obtained from molecular models.

A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full-Length TAR RNA and Investigation by In-Line Probing and EPR Spectroscopy.

EPR studies on RNA are complicated by three major obstacles related to the chemical nature of nitroxide spin labels: Decomposition while oligonucleotides are chemically synthesized, further decay during enzymatic strand ligation, and undetected changes in conformational equilibria due to the steric demand of the label. Herein possible solutions for all three problems are presented: A 2-nitrobenzyloxymethyl protective group for nitroxides that is stable under all conditions of chemical RNA synthesis and can be removed photochemically. By careful selection of ligation sites and splint oligonucleotides, high yields were achieved in the assembly of a full-length HIV-1 TAR RNA labeled with two protected nitroxide groups. PELDOR measurements on spin-labeled TAR in the absence and presence of arginine amide indicated arrest of interhelical motions on ligand binding. Finally, even minor changes in conformation due to the presence of spin labels are detected with high sensitivity by in-line probing.

Peptidomimetics That Inhibit and Partially Reverse the Aggregation of Aß1-42.

The peptide sequence KLVFF resembles the hydrophobic core of the Aß peptide known to form amyloid plaques in Alzheimer's disease. Starting from its retro-inverso peptide, we have synthesized three generations of peptidomimetics. Step by step natural amino acids have been replaced by aromatic building blocks accessible from the Pd-catalyzed Catellani reaction. The final compound 18 is stable against proteolytic decay and largely prevents the aggregation of Aß1-42 over extended periods of time. The activity of the new inhibitors was tested first by fluorescence correlation spectroscopy. For closer examination of compound 18, additional techniques were also applied: laser-induced liquid bead ion desorption mass spectrometry, confocal laser scanning microscopy, thioflavin T fluorescence, and gel electrophoresis. Compound 18 not only retards the aggregation of chemically synthesized Aß but also can partially dissolve the oligomeric structures. Thioflavin binding mature fibrils, however, seem to resist the inhibitor.

A chiral analog of the bicyclic guanidine TBD: synthesis, structure and Brønsted base catalysis.

Starting from (S)-ß-phenylalanine, easily accessible by lipase-catalyzed kinetic resolution, a chiral triamine was assembled by a reductive amination and finally cyclized to form the title compound 10. In the crystals of the guanidinium benzoate salt the six membered rings of 10 adopt conformations close to an envelope with the phenyl substituents in pseudo-axial positions. The unprotonated guanidine 10 catalyzes Diels-Alder reactions of anthrones and maleimides (25-30% ee). It also promotes as a strong Brønsted base the retro-aldol reaction of some cycloadducts with kinetic resolution of the enantiomers. In three cases, the retro-aldol products (48-83% ee) could be recrystallized to high enantiopurity (≥95% ee). The absolute configuration of several compounds is supported by anomalous X-ray diffraction and by chemical correlation.

A Bis(guanidinium)alcohol Attached to a Hairpin Polyamide: Synthesis, DNA Binding, and Plasmid Cleavage.

Bis(guanidinium)alcohols have been designed to react with phosphodiester substrates in a fast transphosphorylation step, a quasi-intramolecular process taking place in contact ion pairs. Here the attachment of such compounds to Dervan-type hairpin polyamides is described. The resulting conjugate 1 binds to AT-rich DNA duplexes with affinity similar to that of the parent polyamide as shown by UV melting experiments and CD titrations. Conjugate 1 nicks plasmid DNA at concentrations ranging from micromolar to high nanomolar.

Studies on Tris(2-aminobenzimidazole)-PNA Based Artificial Nucleases: A Comparison of Two Analytical Techniques.

A new peptide nucleic acid (PNA) construct carrying a tris(2-aminobenzimidazole) phosphodiester cleaver is presented. This non-metal-based artificial nuclease hydrolyzes RNA substrates that form a bulge upon binding to the PNA. Reaction rates depend on the bulge sequence. For conjugates of tris(2-aminobenzimidazole), substrate turnover is shown for the first time. Two methods of analysis for the kinetics are compared: IE-HPLC separation of oligonucleotide fragments and analysis of Cy5-labeled oligonucleotide fragments by denaturating PAGE on a DNA sequencer, respectively. The different methods give rates that are in the same range where, in general, the substrates for the sequencer method give slightly lower rates.

Sequence-specific RNA cleavage by PNA conjugates of the metal-free artificial ribonuclease tris(2-aminobenzimidazole).

Tris(2-aminobenzimidazole) conjugates with antisense oligonucleotides are effective site-specific RNA cleavers. Their mechanism of action is independent of metal ions. Here we investigate conjugates with peptide nucleic acids (PNA). RNA degradation occurs with similar rates and substrate specificities as in experiments with DNA conjugates we performed earlier. Although aggregation phenomena are observed in some cases, proper substrate recognition is not compromised. While our previous synthesis of 2-aminobenzimidazoles required an HgO induced cyclization step, a mercury free variant is described herein.