Nukleotidanaloga als starre Spinmarker für DNA und RNA

Nucleic acids are one of the important classes of biomolecules together with carbohydrates, proteins and lipids. Both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are most well known for their respective roles in the storage and expression of genetic information. Over the course of the last decades, nucleic acids with a variety of other functions have been discovered in biological organisms or created artificially. Examples of these functional nucleic acids are riboswitches, aptamers and ribozymes. In order to gain information regarding their function, several analytical methods can be used. Electron paramagnetic resonance (EPR) spectroscopy is one of several techniques which can be used to study nucleic acid structure and dynamics. However, EPR spectroscopy requires unpaired electrons and because nucleic acids themselves are not paramagnetic, the incorporation of spin labels which carry a radical is necessary. Here, three new spin labels for the analysis of nucleic acids by EPR spectroscopy are presented. All of them share two important design features. First, the paramagnetic center is located at a nitroxide, flanked by ethyl groups to prevent nitroxide degradation, for example during solid phase synthesis. Furthermore, they were designed with rigidity as an important quality, in order to be useful for applications like pulsed electron double resonance (PELDOR) spectroscopy, where independent motion of the spin labels relative to the macromolecule has a noticeable negative effect on the precision of the measurements. Benzi-spin is a spin label which differs from most previous examples of rigid spin labels in that rather than being based on a canonical nucleoside, with a specific base pairing partner, it is supposed to be a universal nucleoside which is sufficiently rigid for EPR measurements when placed opposite to a number of different nucleosides. Benzi-spin was successfully incorporated into a 20 nt oligonucleotide and its base pairing behavior with seven different nucleosides was examined by UV/VIS thermal denaturation and continuous wave (CW) EPR experiments. The results show only minor differences between the different nucleosides, thus confirming the ability of benzi-spin to act as a universally applicable spin label. Lumi-spin is derived from lumichrome. It features a rigid scaffold, as well as a free 2'-hydroxy group, which should make it well suited for PELDOR experiments once it is incorporated into RNA oligonucleotides. EÇr is based on the Ç family of spin labels, which contains the most well known rigid spin labels for nucleic acids to this day. It is essentially a version of EÇm with a free 2'-hydroxy group. It was converted to triphosphate EÇrTP and used for primer extension experiments to test the viability of enzymatic incorporation of rigid spin labels into oligonucleotides as an alternative to solid-phase synthesis. Incorporation into DNA by Therminator III DNA polymerase in both single-nucleotide and full-length primer extensions was achieved. All three of these spin labels represent further additions to the expanding toolbox of EPR spectroscopy on nucleic acids and might prove valuable for future research.Nukleinsäuren sind neben den Kohlenhydraten, Proteinen und Lipiden eine der wichtigen Klassen von Biomolekülen. Sowohl Deoxyribonukleinsäure (DNA) und Ribonukleinsäure (RNA) sind am besten für ihre Funktionen bei der Speicherung und Expression der genetischen Informationen bekannt. Während der letzten Jahrzehnte wurden Nukleinsäuren mit einer Vielzahl von Funktionen in biologischen Organismen entdeckt oder künstlich hergestellt. Beispiele für diese funktionellen Nukleinsäuren sind Riboswitches, Aptamere und Ribozyme. Um Informationen über ihre Funktionsweisen zu erhalten, können verschiedene analytische Methoden verwendet werden. Elektronenspinresonanzspektroscopie (ESR) ist eine Analysetechnik, die Aufschluss über Struktur und Dynamik von Nukleinsäuren geben kann. Für ESR Messungen werden ungepaarte Elektronen benötigt, sodass nicht paramagnetische Verbindungen mit einem Spinmarker modifiziert werden müssen, der ein Radikal trägt. In dieser Arbeit werden drei neue Spinmarker für die ESR Analyse von Nukleinsäuren vorgestellt. Allen liegen zwei Designprinzipien zugrunde. Erstens wird als paramagnetische Verbindung ein Nitroxid verwendet, welches von Ethylgruppen flankiert wird um das Radikal zu stabilisieren, zum Beispiel gegen Reagenzien, die in der Festphasensynthese verwendet werden. Zweitens sind die Nitroxide Teil starrer Ringsysteme. Dies ist besonders wichtig für Anwendungen wie Abstandsmessungen mittels Pulselektronendoppelresonanzspektroskopie (PELDOR), wo die Genauigkeit der Messung von Bewegungen der Spinmarker relativ zum Makromolekül beeinträchtigt wird. Benzi-spin unterscheidet sich von vielen anderen starren Spinmarkern dadurch, dass es nicht auf einem kanonischen Nukleosid mit einem spezifischen Bindungspartner basiert. Stattdessen handelt es sich um ein universelles Nukleosid, das unabhängig vom gegenüberliegenden Nukleosid starr genug für ESR Messungen ist. Benzi-spin wurde erfolgreich in ein Oligonucleotid eingebaut und seine Basenpaarung mit sieben verschiedenen Nukleosiden mittels UV/VIS Schmelzkurven und Continuous Wave (CW) ESR Experimenten untersucht. Die Ergebnisse zeigen nur geringe Unterschiede zwischen den verschiedenen Nukleosiden, was die Einsetzbarkeit von Benzi-spin als universeller Spinmarker bestätigt. Lumi-spin ist vom Lumichrom abgeleitet. Es zeichnet sich durch ein starres Gerüst und eine freie 2'-Hydroxygruppe aus, wodurch es gut für PELDOR Messungen in RNA geeignet sein sollte. EÇr gehört zur Ç Familie, welche die am besten bekannten starren Spinmarker für Nukleinsäuren enthält. Es handelt sich um eine Version von EÇm mit einer freien 2'-Hydroxygruppe. EÇr wurde zum Triphosphat EÇrTP konvertiert und für Primer Extension Experimente verwendet um die Möglichkeit des enzymatischen Einbaus starrer Spinmarker in Oligonukleotide als Alternative zur Festphasensynthese zu prüfen. Der Einbau in DNA mit Therminator III DNA Polymerase in Primer Extensions war erfolgreich. Alle drei Spinmarker erweitern die Möglichkeiten der ESR-spektroskopischen Untersuchung von Nukleinsäuren und können sich für zukünftige Forschung als nützlich erweisen

Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryoelectron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication

USING NONCONTINGENT REINFORCEMENT TO INCREASE COMPLIANCE WITH WEARING PRESCRIPTION PROSTHESES

We evaluated the effects of noncontingent reinforcement (NCR) on compliance with wearing foot orthotics and a hearing aid with 2 individuals. Results showed that NCR increased the participants' compliance with wearing prescription prostheses to 100% after just a few 5-min sessions, and the behavior change was maintained during lengthier sessions. The results are discussed in terms of the potential value-altering effects of NCR

Translation of non-standard codon nucleotides reveals minimal requirements for codon-anticodon interactions

International audienc

Specific labeling of peptidoglycan precursors as a tool for bacterial cell wall studies

Wall chart: The predominant component of the bacterial cell wall, peptidoglycan, consists of long alternating stretches of aminosugar subunits interlinked in a large three-dimensional network and is formed from precursors through several cytosolic and membrane-bound steps. The high tolerance of the cell wall synthesis machinery allows for the use of labeled precursor derivatives to study diverse aspects of bacterial cell wall synthesis and interaction with antibiotics. Because of its importance for bacterial cell survival, the bacterial cell wall is an attractive target for new antibiotics in a time of great demand for new antibiotic compounds. Therefore, more knowledge about the diverse processes related to bacterial cell wall synthesis is needed. The cell wall is located on the exterior of the cell and consists mainly of peptidoglycan, a large macromolecule built up from a three-dimensional network of aminosugar strands interlinked with peptide bridges. The subunits of peptidoglycan are synthesized inside the cell before they are transported to the exterior in order to be incorporated into the growing peptidoglycan. The high flexibility of the cell wall synthesis machinery towards unnatural derivatives of these subunits enables research on the bacterial cell wall using labeled compounds. This review highlights the high potential of labeled cell wall precursors in various areas of cell wall research. Labeled precursors can be used in investigating direct cell wall-antibiotic interactions and in cell wall synthesis and localization studies. Moreover, these compounds can provide a powerful tool in the elucidation of the cell wall proteome, the wallosome, and thus, might provide new targets for antibiotic

Universal Dependencies 2.11

Universal Dependencies is a project that seeks to develop cross-linguistically consistent treebank annotation for many languages, with the goal of facilitating multilingual parser development, cross-lingual learning, and parsing research from a language typology perspective. The annotation scheme is based on (universal) Stanford dependencies (de Marneffe et al., 2006, 2008, 2014), Google universal part-of-speech tags (Petrov et al., 2012), and the Interset interlingua for morphosyntactic tagsets (Zeman, 2008)

Universal Dependencies 2.3

Universal Dependencies is a project that seeks to develop cross-linguistically consistent treebank annotation for many languages, with the goal of facilitating multilingual parser development, cross-lingual learning, and parsing research from a language typology perspective. The annotation scheme is based on (universal) Stanford dependencies (de Marneffe et al., 2006, 2008, 2014), Google universal part-of-speech tags (Petrov et al., 2012), and the Interset interlingua for morphosyntactic tagsets (Zeman, 2008)

Universal Dependencies 2.8.1

Universal Dependencies is a project that seeks to develop cross-linguistically consistent treebank annotation for many languages, with the goal of facilitating multilingual parser development, cross-lingual learning, and parsing research from a language typology perspective. The annotation scheme is based on (universal) Stanford dependencies (de Marneffe et al., 2006, 2008, 2014), Google universal part-of-speech tags (Petrov et al., 2012), and the Interset interlingua for morphosyntactic tagsets (Zeman, 2008). Version 2.8.1 fixes a bug in 2.8 where a portion of the Dutch Alpino treebank was accidentally omitted