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Recent advances in the development of immunoassays and nucleic acid assays have improved the performance and increased the sensitivity of sensors that are based on biochemical recognition. The new approaches taken by researchers include detecting pathogens by detecting their nucleic acids, using new nontoxic reporter entities for generating signals, and downscaling and miniaturizing sensors to micromigration and microfluidic formats. This dissertation connects some of these successful approaches, thereby leading to the development of novel nucleic acid sensors for rapid and easy detection of pathogens. The author's goal was to develop diagnostic tools that enable investigators to detect pathogens rapidly and on site. While the sensors can be used to detect any pathogen, the author first customized them for detecting particularly Cryptosporidium parvum, a pathogen whose detection is important, yet presents many challenges. Chapter 2 of this thesis presents a novel test-strip for the detection of C. parvum. The test-strip is designed to detect nucleic acids rather than proteins or other epitopes. While test strips are commonly used for sensors based on immunological recognition, this format is very new in applications in which nucleic acids are detected. Further, to indicate the presence or absence of a specific target on the test strip, dye-entrapped, oligonucleotide-tagged liposomes are employed. Using liposomes as reporter particles has advantages over using other reporter labels, because the cavity that the phospholipidic membranes of the liposomes form can be filled with up to 106 dye molecules. By using heterobifunctional linkers liposomes can be tagged with oligonucleotides, thereby enabling their use in nucleic acid hybridization assays. The developed test-strip provides an internal control. The limit of detection is 2.7 fmol/mL with a sample volume of 30 mL. In chapter 3 the detection of nucleic acids by means of oligonucleotide-tagged liposomes is scaled down to a microfluidic assay format. Because the application of biosensors to microfluidic formats is very new in the field of analytical chemistry, the first part of this chapter is devoted to developing the design and the method to fabricate the microchip devices. The performance of the microchips is then optimized by investigating the interactions of nucleic acids and liposomes with the material the chips consist of and by passivating the surface of the chips with blocking reagents. The developed microfluidic chip enabled us to reduce the sample volume needed for one assay to 12.5 mL. The limit of detection of this assay was determined to be 0.4 fmol/mL. Chapters 4 and 5 expand on the development of the microfluidic assay. A prototype microfluidic array that is able to detect multiple analytes in a single sample simultaneously is developed. Using such an array will enable investigators to detect pathogens that occur in the same environment, for example, C. parvum and Giardia duodenalis by conducting a single test. The array's ability to perform multiple sample analysis is shown by detecting different concentrations of target nucleic acids. Further, the author developed a microfluidic chip in which interdigitated microelectrode arrays (IDAs) that consist of closely spaced microelectrodes are integrated. The IDAs facilitate electrochemical detection of cryptosporidial RNA. Electrochemical detection schemes offer benefits of technical simplicity, speed, and sensitivity. In this project liposomes are filled with electrochemically active molecules and are then utilized to generate electrochemical signals. Chapter 6 explores the feasibility of liposomes for enhancing signals derived from nucleic acid hybridization in surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy offers advantages because nucleic acid hybridization can be monitored in real time and under homogeneous conditions because no washing steps are required. SPR spectroscopy is very sensitive and it can be expected that, in the future, SPR will be integrated into microfluidic nucleic acid sensors.
Humane Coronaviren sind wichtige Pathogene, die vor allem mit respiratorischen (z.B. SARS) und enteralen Erkrankungen assoziiert sind. Coronaviren besitzen das größte gegenwärtig bekannte RNA-Genom aller Viren (ca. 30 Kilobasen). Die Replikation des Genoms und die Synthese zahlreicher subgenomischer RNAs, die die viralen Strukturproteine und einige akzessorische, vermutlich virulenzassoziierte, Proteine kodieren, erfolgt durch die virale Replikase. Die coronavirale Replikase ist ein Multienzym-Komplex, der durch die proteolytische Prozessierung großer Vorläuferproteine (Polyproteine pp1a und pp1ab) entsteht und 16 virale Nichtstrukturproteine (nsp), aber auch einige zelluläre Proteine, beinhaltet. Obwohl die Charakterisierung der Funktionen der einzelnen Proteine und das Verständnis der molekularen Grundlagen der coronaviralen Replikation noch in ihren Anfängen stecken, ist bereits jetzt klar, dass die an der Replikation beteiligten Mechanismen deutlich komplexer sind als bei den meisten anderen RNA-Viren. Man hofft, dass aus der Untersuchung der einzelnen an der Replikation beteiligten Proteine Erkenntnisse zu den Besonderheiten des Lebenszyklus dieser ungewöhnlich großen RNA-Viren abgeleitet werden können und dass sich daraus auch Ansatzpunkte für die Entwicklung von Inhibitoren einzelner Proteine/Enzyme ergeben, die für eine zukünftige antivirale Therapie genutzt werden könnten. In der vorliegenden Arbeit wurden zwei enzymatische Aktivitäten von Coronaviren, eine Helikase und eine Endonuklease, die Teil der coronaviralen Nichtstrukturproteine nsp13 bzw. nsp15 sind, in vitro untersucht. Zur Etablierung allgemeingültiger Prinzipien coronaviraler Enzymaktivitäten wurden die homologen Proteine von HCoV-229E und SARS-CoV, also von Vertretern unterschiedlicher serologischer und genetischer Coronavirus-Gruppen, parallel untersucht und ihre Eigenschaften miteinander verglichen. Die nsp13-Helikase des SARSCoronavirus wurde als bakterielles Fusionsprotein exprimiert, und die nsp13-Helikase des humanen Coronavirus 229E wurde in Insektenzellen mittels baculoviraler Vektoren exprimiert. Beide Proteine zeigten Polynukleotid-stimulierbare NTPase- und 5'-3'-Helikase-Aktivitäten. Darüber hinaus besaßen sie vergleichbare Hydrolyseaktivitäten gegenüber den 8 getesteten Ribound Desoxyribonukleosidtriphosphaten. Die Anwesenheit von poly(U) führte zu einer 3-fachen Erhöhung der katalytischen Effizienz (kcat/Km) und einer etwa 100-fachen Steigerung der Hydrolysegeschwindigkeit (kcat). Es wurde am Beispiel von HCoV-229E-nsp13 gezeigt, dass Nukleinsäuresubstrate mit hoher Affinität (K50 ≈ 10-8 M), jedoch ohne erkennbare Präferenz für einzel- oder doppelsträngige DNA- oder RNA-Substrate gebunden werden. Solch eine feste Bindung ist typisch für Enzyme, die prozessiv mit Nukleinsäuren interagieren. Sie korreliert darüber hinaus mit der beobachteten effizienten Entwindung (Trennung) von RNA- und DNADuplexen mit langen, doppelsträngigen Bereichen von 500 Basenpaaren und mehr. Dies legt eine Funktion als replikative Helikase nahe, wie sie beispielweise bei der effektiven Entwindung doppelsträngiger replikativer Intermediate benötigt werden könnte. In dieser Arbeit wurde darüber hinaus eine neue enzymatische Aktivität coronaviraler Helikasen entdeckt. Die gefundene RNA-5'-Triphosphatase-Aktivität nutzt das aktive Zentrum der NTPase-Aktivität und katalysiert wahrscheinlich die erste Reaktion innerhalb der Synthese der Cap-Struktur am 5’- Ende viraler RNAs. Die sehr ähnlichen biochemischen Eigenschaften der HCoV-229E- und SARS-CoV-Helikasen lassen vermuten, dass die Enzymologie der viralen RNA-Synthese (trotz relativ geringer Sequenzidentität der beteiligten Enzyme) unter den Vertretern unterschiedlicher Gruppen von Coronaviren konserviert ist. Der zweite Teil der Arbeit beschäftigte sich mit der biochemischen Charakterisierung des Nichtstrukturproteins nsp15, für das eine Endonuklease-Aktivität vorhergesagt worden war. Auch in diesem Fall wurden die entsprechenden Proteine von HCoV-229E und SARS-CoV charakterisiert. Beide (bakteriell exprimierten) Enzyme zeigten identische enzymatische Eigenschaften. In-vitro-Experimente bestätigten, dass diese Proteine eine Mn2+-abhängige RNA- (jedoch nicht DNA-) Endonukleaseaktivität besitzen. Sie spalten doppelsträngige RNA deutlich effektiver und spezifischer als einzelsträngige RNA. Die Enzyme spalten an Uridylat-Resten und erzeugen Produkte mit 2', 3'-Zyklophosphat-Enden. Bei doppelsträngigen RNA-Substraten wurde eine Spezifität für 5'-GU(U)-3' gefunden. Die Tatsache, dass diese Sequenz in den nidoviralen transkriptionsregulierenden Sequenzen (TRS) der Minusstränge konserviert ist und auch die Endonuklease bei allen Nidoviren konserviert ist, unterstützt die Hypothese, dass die Endonukleaseaktivität eine spezifische Funktion innerhalb der coronaviralen (nidoviralen) diskontinuierlichen Transkription besitzt.
Immunotherapeutic strategies may be a treatment option in patients with refractory acute myelogenous leukemia (AML) or, in cases of complete remission after conventional therapy regimens, may help to reduce disease recurrence or delay time to progression. Evidence suggests a key role of dendritic cells (DCs) in cancer immunotherapy due to their capacity to present tumour antigens to effector cells. We generated cytokine-induced killer (CIK) cells from healthy donors and examined their responses in vitro in an LDH release assay against three cell lines and allogeneic HLA non-matched blasts from three patients with de novo AML after coincubation with autologous peripheral blood monocyte-derived DCs. Although DCs were unable to enhance CIK cell effects against all three cell lines tested, the cytotoxic activity against the patients’ AML cells increased after coculture with mature DCs, which was significant in two of three patients. However, neither prior pulsing of the DCs with blast cell lysates nor with leukemic cell-derived total RNA further enhanced the lytic capacity of the CIK cells. On the contrary, pulsing reduced or even reversed the cytotoxic activity of the effector cells. This decrease of allogeneic cytotoxicity led us to conclude that monocyte-derived DCs may be useful in autologous or allogeneic vaccine strategies for the treatment of AML or in priming donor lymphocytes in vitro, but unfractionated antigens as pulsing agents may have inhibitory effects on T cell efficiency and their employment in immunotherapeutic strategies for AML seems questionable.
The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is an important model to elucidate the mechanisms involved in the interaction with the host. To gain insight into the transcriptome of the Xcv strain 85-10, we took a differential RNA sequencing (dRNA-seq) approach. Using a novel method to automatically generate comprehensive transcription start site (TSS) maps we report 1421 putative TSSs in the Xcv genome. Genes in Xcv exhibit a poorly conserved -10 promoter element and no consensus Shine-Dalgarno sequence. Moreover, 14% of all mRNAs are leaderless and 13% of them have unusually long 5'-UTRs. Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv. Expression of eight intergenic transcripts was controlled by HrpG and HrpX, key regulators of the Xcv type III secretion system. More detailed characterization identified sX12 as a small RNA that controls virulence of Xcv by affecting the interaction of the pathogen and its host plants. The transcriptional landscape of Xcv is unexpectedly complex, featuring abundant antisense transcripts, alternative TSSs and clade-specific small RNAs.
Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade \(Milnesium\) \(tardigradum\) were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from \(Hypsibius\) \(dujardini\), revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for \(M.\) \(tardigradum\) are different from typical motifs known from higher animals. \(M.\) \(tardigradum\) and \(H.\) \(dujardini\) protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of \(M.\) \(tardigradum\). These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and \(M.\) \(tardigradum\) in particular so highly stress resistant.
MicroRNAs (miRNAs) play regulatory roles in diverse processes in both eukaryotic hosts and their viruses, yet fundamental questions remain about which viruses code for miRNAs and the functions that they serve. Simian foamy viruses (SFVs) of Old World monkeys and apes can zoonotically infect humans and, by ill-defined mechanisms, take up lifelong infections in their hosts. Here, we report that SFVs encode multiple miRNAs via a noncanonical mode of biogenesis. The primary SFV miRNA transcripts (pri-miRNAs) are transcribed by RNA polymerase III (RNAP III) and take multiple forms, including some that are cleaved by Drosha. However, these miRNAs are generated in a context-dependent fashion, as longer RNAP II transcripts spanning this region are resistant to Drosha cleavage. This suggests that the virus may avoid any fitness penalty that could be associated with viral genome/transcript cleavage. Two SFV miRNAs share sequence similarity and functionality with notable host miRNAs, the lymphoproliferative miRNA miR-155 and the innate immunity suppressor miR-132. These results have important implications regarding foamy virus biology, viral miRNAs, and the development of retroviral-based vectors. IMPORTANCE Fundamental questions remain about which viruses encode miRNAs and their associated functions. Currently, few natural viruses with RNA genomes have been reported to encode miRNAs. Simian foamy viruses are retroviruses that are prevalent in nonhuman host populations, and some can zoonotically infect humans who hunt primates or work as animal caretakers. We identify a cluster of miRNAs encoded by SFV. Characterization of these miRNAs reveals evolutionarily conserved, unconventional mechanisms to generate small RNAs. Several SFV miRNAs share sequence similarity and functionality with host miRNAs, including the oncogenic miRNA miR-155 and innate immunity suppressor miR-132. Strikingly, unrelated herpesviruses also tap into one or both of these same regulatory pathways, implying relevance to a broad range of viruses. These findings provide new insights with respect to foamy virus biology and vectorology.
Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts.
Epitranscriptome modifications are required for structure and function of RNA and defects in these pathways have been associated with human disease. Here we identify the RNA target for the previously uncharacterized 5-methylcytosine (m5C) methyltransferase NSun3 and link m5C RNA modifications with energy metabolism. Using whole-exome sequencing, we identified loss-of-function mutations in NSUN3 in a patient presenting with combined mitochondrial respiratory chain complex deficiency. Patient-derived fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of NSun3. We show that NSun3 is required for deposition of m5C at the anticodon loop in the mitochondrially encoded transfer RNA methionine (mt-tRNAMet). Further, we demonstrate that m5C deficiency in mt-tRNAMet results in the lack of 5-formylcytosine (f5C) at the same tRNA position. Our findings demonstrate that NSUN3 is necessary for efficient mitochondrial translation and reveal that f5C in human mitochondrial RNA is generated by oxidative processing of m5C.
The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq
(2017)
Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical σ70-type promoters, suggesting the existence of activators that compensate for the lack of −35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.
Fluorogenic Aptamers and Fluorescent Nucleoside Analogs as Probes for RNA Structure and Function
(2020)
RNA plays a key role in numerous cellular processes beyond the central dogma of molecular biology. Observing and understanding this wealth of functions, discovering new ones and engineering them into purpose-built tools requires a sensitive means of observation. Over the past decade, fluorogenic aptamers have emerged to fill this niche. These short oligonucleotides are generated by in vitro selection to specifically interact with small organic fluorophores and can be utilized as genetically encoded tags for RNAs of interest.
The most versatile class of fluorogenic aptamers is based on derivatives of hydroxybenzylidene imidazolone (HBI), a conditional fluorophore mimicking the chromophore structure found in green and red fluorescent proteins. The respective aptamers are well-known by the “vegetable” nomenclature, including Spinach, Broccoli and Corn, and have found numerous applications for studying RNA function in vitro and in cells.
Their success, however, is somewhat overshadowed by individual shortcomings such as a propensity for misfolding, dependence on unphysiologically high concentrations of magnesium ions or, in the case of Corn, dimerization that might affect the function of the tagged RNA. Moreover, most fluorogenic aptamers exhibit limited ligand promiscuity by design, thereby restricting their potential for spectral tuning to a narrow window of wavelengths.
This thesis details the characterization of a new fluorogenic aptamer system nicknamed Chili. Chili is derived from an aptamer that was originally selected to bind 4-hydroxy-3,5-dimethoxy¬hydroxy-benzylidene imidazolone (DMHBI), resulting in a green fluorescent complex. Unlike other aptamers of its kind, Chili engages in a proton transfer cycle with the bound ligand, resulting in a remarkably large Stokes shift of more than 130 nm.
By means of an empirical ligand optimization approach, several new DMHBI derivatives were found that bind to Chili with high affinity, furnishing complexes up to 7.5 times brighter compared to the parent ligand. In addition, Chili binds to π-extended DMHBI derivatives that confer fluorescence in the yellow–red region of the visible spectrum. The highest affinity and degree of fluorescence turn-on for both green and red fluorogenic ligands were achieved by the incorporation of a unique, positively charged substituent into the HBI scaffold.
Supplemented by NMR spectroscopy, kinetic and thermodynamic studies showed that the binding site of Chili is loosely preorganized in the absence of ligand and likely forms a G-quadruplex upon ligand binding.
To showcase future applications, Chili was incorporated into a FRET sensor for monitoring the cleavage of an RNA substrate by a 10-23 DNAzyme.
Besides aptamers as macromolecular fluorescent complexes, fluorescent nucleobase analogs are powerful small isomorphic components of RNA suitable for studying structure and folding. Here, the highly emissive nucleobase analog 4-cyanoindole (4CI) was developed into a ribonucleoside (r4CI) for this purpose. A new phosphoramidite building block was synthesized to enable site-specific incorporation of 4CI into RNA.
Thermal denaturation experiments confirmed that 4CI behaves as a universal nucleobase, i.e. without bias towards any particular hybridization partner. Photophysical characterization established r4CI as a generally useful fluorescent ribonucleoside analog. In this work, it was employed to gain further insight into the structure of the Chili aptamer. Using several 4CI-modified Chili–HBI complexes, a novel base–ligand FRET assay was established to obtain a set of combined distance and orientation restraints for the tertiary structure of the aptamer.
In addition to their utility for interrogating structure and binding, supramolecular FRET pairs comprising a fluorescent nucleobase analog donor and an innately fluorogenic acceptor hold great promise for the construction of color-switchable RNA aptamer sensor devices.