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Structural transitions of RNA between alternate conformations with similar stabilities are associated with important aspects of cellular function. Few techniques presently exist that are capable of monitoring such transitions and thereby provide insight into RNA dynamics and function at atomic resolution. Riboswitches are found in the 5'-UTR of mRNA and control gene expression through structural transitions after ligand recognition. A time-resolved NMR strategy was established in conjunction with laser-triggered release of the ligand from a photocaged derivative in situ to monitor the hypoxanthine-induced folding of the guanine-sensing riboswitch aptamer domain of the Bacillus subtilis xpt-pbuX operon at atomic resolution. Combining selective isotope labeling of the RNA with NMR filter techniques resulted in significant spectral resolution and allowed kinetic analysis of the buildup rates for individual nucleotides in real time. Three distinct kinetic steps associated with the ligand-induced folding were delineated. After initial complex encounter the ligand-binding pocket is formed and results in subsequent stabilization of a remote long-range loop-loop interaction. Incorporation of NMR data into experimentally restrained molecular dynamics simulations provided insight into the RNA structural ensembles involved during the conformational transition.

In bacteria, the regulation of gene expression by cis-acting transcriptional riboswitches located in the 5'-untranslated regions of messenger RNA requires the temporal synchronization of RNA synthesis and ligand binding-dependent conformational refolding. Ligand binding to the aptamer domain of the riboswitch induces premature termination of the mRNA synthesis of ligand-associated genes due to the coupled formation of 3'-structural elements acting as terminators. To date, there has been no high resolution structural description of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA element. Here, we show that for the guanine-sensing xpt-pbuX riboswitch from Bacillus subtilis, the conformation of the full-length transcripts is static: it exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand. We show that only the combined matching of transcription rates and ligand binding enables transcription intermediates to undergo ligand-dependent conformational refolding.

The development of a nanoparticle RNA vaccine is reported that preferentially targets dendritic cells after systemic administration, and is shown to provide durable interferon-α-dependent antigen-specific immunity in mouse tumour models; initial results in advanced melanoma patients indicate potential efficacy in humans. An anti-cancer nanoparticulate RNA vaccine The systemic delivery of vaccine antigens into the dendritic or antigen-presenting cells of the immune system faces many technical challenges. This study reports the development of a nanoparticle RNA vaccine that preferentially targets dendritic cells after systemic administration. The vaccine consists of RNA-lipoplexes based on well-known lipid carriers; targeting is achieved by optimally adjusting the negative net charge of the nanoparticles, with no need for functionalization with molecular ligands. Intravenous administration produces durable type-I-interferon-dependent antigen-specific immunity in mouse tumour models. Initial results in patients with advanced melanoma indicate potential efficacy in humans. Virtually any tumour antigen can be encoded by RNA, so this approach is potentially more generally applicable in cancer immunotherapy. Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses 1 . However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated 2 . We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA 3 , 4 , RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.

The authors report the first-in-human application of personalized neo-antigen RNA vaccines in patients with melanoma. Personalized cancer vaccine trials Neoantigens have long been considered optimal targets for anti-tumour vaccines, and recent mutation coding and prediction techniques have aimed to streamline their identification and selection. Two papers in this issue report results from personalized neoantigen vaccine trials in patients with cancer. Catherine Wu and colleagues report the results of a phase I trial of a personalized cancer vaccine that targets up to 20 patient neoantigens. The vaccine was safe and induced tumour-antigen-specific immune responses. Four out of six patients treated showed no recurrence at 25 months, and progressing patients responded to further therapy with checkpoint inhibitor. Ugur Sahin and colleagues report the first-in-human application of a personalized neoantigen vaccine in patients with melanoma. Their vaccination strategy includes sequencing and computational identification of neoantigens from patients, and design and manufacture of a poly-antigen RNA vaccine for treatment. In 13 patients, the vaccine boosted immunity against some of the selected tumour antigens from the individual patients, and two patients showed infiltration of tumour-reactive T cells. These results suggest that personalized vaccines could be refined and tailored to provide clinical benefit as cancer immunotherapies. T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations 1 , 2 . Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of β2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.

We present here a set of ¹³C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose ¹³C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4′ nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1′,H1′ ribose signals. The experiments were applied to two RNA hairpin structures. The current set of ¹³C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, ¹³C-direct detected NMR methods constitute useful complements to the conventional ¹H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.

We present here a set of 13 C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose 13 C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4′ nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1′,H1′ ribose signals. The experiments were applied to two RNA hairpin structures. The current set of 13 C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, 13 C-direct detected NMR methods constitute useful complements to the conventional 1 H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.

We present here a set of ^sup 13^C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose ^sup 13^C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4' nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1',H1' ribose signals. The experiments were applied to two RNA hairpin structures. The current set of ^sup 13^C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, ^sup 13^C-direct detected NMR methods constitute useful complements to the conventional ^sup 1^H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.[PUBLICATION ABSTRACT]

Obwohl zahlreiche zelluläre Funktionen von RNAs in direktem Zusammenhang mit Proteinen stehen, wurde auch eine Vielzahl von, unter anderem regulatorischen, RNA-Motiven identifiziert, die ihre Funktion ohne eine initiale Beteiligung von Proteinen ausüben. Das detaillierte Verständnis der zu Grunde liegenden Regulationsmechanismen beinhaltet die Charakterisierung von beteiligten RNA-Architekturen und deren funktionaler Stabilitäten, von dynamischen Aspekten der RNA-Faltungsprozesse sowie die Korrelation dieser Charakteristika mit zellulären Funktionen. Im Rahmen dieser Arbeit wurden strukturelle, thermodynamische und kinetische Aspekte der Ligand-bindenden Guanin Riboswitch-RNA Aptamerdomäne des xpt-pbuX Operons aus B. subtilis und eines Cofaktor-abhängigen katalytischen RNA-Motivs, des ‛Adenin-abhängigen Hairpin Ribozyms’, untersucht.Dem Modell der durch Riboswitch-RNA Elemente vermittelten Genregulation zufolge werden in Abhängigkeit der Konzentration eines jeweils spezifischen Liganden alternative RNAKonformationen der Ligand-Bindungsdomäne (Aptamerdomäne) stabilisiert, welche die Ausbildung eines 3’-nachfolgenden Strukturelementes und infolge die Genexpression beeinflussen. Die Untersuchung der Ligand-induzierten Faltungsprozesse der Guanin Riboswitch Aptamerdomäne wurde durch die Licht-induzierte Aktivierung des chemisch synthetisierten, photogeschützten Liganden und anschließender Detektion mittels zeitaufgelöster NMR-Methoden erreicht. Um bei der Größe der untersuchten RNA die Analyse der Geschwindigkeitskonstanten individueller Signale zu ermöglichen, wurde eine Kombination aus selektiver Isotopenmarkierung und NMR-Filtermethoden entwickelt. Auf der Basis der Analyse von NMR-Linienbreiten wurde zusätzlich eine schwach affine und nicht-spezifische Ligandbindung der Riboswitch-RNA postuliert. Das Einbeziehen der NMR-spektroskopischen Daten in MD-Simulationen ermöglichte einen Einblick in denkbare, in den Ligand-Bindungsprozess involvierte RNA-Strukturensembles und damit die Beschreibung eines Modells dieses Ligandinduzierten RNA-Faltungsprozesses. Mittels NMR-spektroskopischer und biophysikalischer Methoden wurde der Einfluss von RNA-Mg2+-Interaktionen auf die RNA-Konformationen der Guanin Riboswitch Aptamerdomäne (Gswapt) und der G37A/C61U-Mutante (Gswloop) im Ligand-freien und -gebundenen Zustand und auf die Stabilitäten individueller Strukturelemente untersucht. Basierend auf diesen Ergebnissen wurden die Funktion des interhelikalen Tertiärstrukturelementes, die Verknüpfung der Tertiärstrukturausbildung mit LigandBindungscharakteristika und die funktionalen Stabilitäten der Riboswitch-RNAs analysiert. RNA-Mg2+-Interaktionen beeinflussen zum einen wesentlich die Temperaturabhängigkeit der funktionalen Stabilitäten von Gswapt und Gswloop. Zum anderen variiert die konformationelle Dynamik der Gswloop-RNA im freien Zustand in Abhängigkeit der Mg2+-Konzentration, was infolge die Eigenschaften der Gswloop-Ligand Komplexbildung beeinflusst. Dies betrifft sowohl nicht-spezifische Ligand-Bindungsprozesse als auch die spezifische RNA-Ligand Komplexbildung. Durch die Nukleotid-spezifische Auflösung der zeitaufgelösten NMR-Kinetiken konnte sowohl die Cofaktor-Abhängigkeit der Strukturausbildung der Ligand-Bindungstasche als auch der Strukturveränderungen entfernt lokalisierter Tertiärstrukturbereiche differenziert analysiert werden. Die unterschiedlichen Charakteristika der RNA-Ensembles der Ausgangsund Endzustände der RNA-Faltungsprozesse ermöglichte die Analyse der Zusammenhänge zwischen Ladungskompensation, Tertiärstrukturausbildung und konformationeller Dynamik und der spezifischen Ligand-induzierten RNA-Faltungsprozesse. Die zeitaufgelösten NMRUntersuchungen der Faltungsprozesse der Guanin Riboswitch Aptamerdomänen und deren Mg2+-Abhängigkeiten zeigten, dass die Geschwindigkeit des Ligand-Bindungsprozesses sensitiv reguliert und wesentlich von strukturellen und Kationen-induzierten Eigenschaften des RNAEnsembles der Ligand-freien Konformation abhängig ist. Die Ausbildung des interhelikalen Tertiärstrukturelementes und RNA-Mg2+-Interaktionen beeinflussen nicht nur die Stabilität der RNA-Struktur, sondern auch die Kinetik des Ligand-Bindungsprozesses.Das ‛Adenin-abhängige Hairpin Ribozym’-Motiv stellt ein Modellsystem eines Ribozyms dar, bei dem katalytische Aktivität durch den exogenen Liganden Adenin induziert wird. Das Verständnis dessen Katalyse erfordert daher die Beantwortung der Fragen, wie die katalytische Aktivität, d. h. die hydrolytische Spaltung des RNA-Phosphatrückgrats, im Detail verwirklicht wird und welche strukturellen Konformationsänderungen mit dem katalytischen Spaltprozess verbunden sind. Mittels NMR-spektroskopischer Methoden wurden die RNA-Konformationen vor und nach der katalytischen Spaltreaktion charakterisiert.

Zusammenfassung NMR-spektroskopische Untersuchungen an regulatorischen RNAs geben Aufschluss über den mechanistischen Zusammenhang von Faltungswegen, dreidimensionaler Struktur, Dynamik und Funktion in Genregulationsprozessen.

The discovery of many novel biological functions for RNA and the realization that many functional RNAs adopt highly intricate three‐dimensional structures have led to a growing interest in exploiting RNA as a drug target. NMR spectroscopy in solution has emerged as a highly versatile technique for characterizing the interaction of RNAs with small molecules for a wide range of affinities at different levels of detail ranging from simple detection of binding events in library screening experiments to a full high‐resolution structural characterization of RNA–small‐molecule complex structures. The NMR spectroscopic techniques that are employed to investigate RNA–small‐molecule interactions are very similar to those developed for proteins. However, RNA has a number of biophysical and NMR spectroscopic characteristics that are distinctly different from those of proteins. Accordingly, on one hand, experiments developed for proteins need to be carefully adapted to the special properties of RNA molecules. On the other hand, RNAs offer unique possibilities that sometimes even simplify the experiments for investigating RNA–ligand interactions. This chapter describes the features that render RNAs especially suitable for studying macromolecule–ligand interactions as well as giving a rationale for the required adaptations in experiments originally developed for protein applications, and describes the most popular approaches in detail using examples from our own work in the field of RNA–ligand interactions.