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Grant
Agency: Cordis | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2013-ITN | Award Amount: 3.47M | Year: 2013

Non-coding RNA (ncRNA) is a new research field in rapid development. It holds the potential to explain many fundamental biological phenomena and there is a vast prospective for the development of ncRNA-derived diagnostic and therapeutic tools. Hence, biotech and pharmaceutical companies are actively looking into this unexplored territory for novel targets. Therefore, there is a substantial and unmet need for the training of scientists in ncRNA biology, methodology and exploitation. The regions encompassing protein coding potential (exons) in humans only amount to 2% of the genome. New sequencing techniques have evidenced that mammalian genomes are pervasively transcribed and have revealed the existence of multiple classes of ncRNAs. Although our knowledge on the multitude of transcripts produced by the non-coding 98% of the genome is still very sketchy, pivotal roles have been established for ncRNAs in organismal development and homeostasis, in cellular proliferation, differentiation and apoptosis and in a broad range of human pathologies. Hence, there is a need to educate young scientists in this emerging and important research field. Aside from increasing our collective understanding of essential biological phenomena, ncRNA also constitute a vast and largely unexplored territory for the development of novel therapeutics and diagnostics. Accordingly, we propose to form a European RNA training network, RNATRAIN. This network will be devoted to educating the next generation of European researchers focusing on the functions and importance of ncRNAs in multidisciplinary projects in which the ncRNAs are studied in the context of development, differentiation and disease. Towards this, a group of 9 top-quality European research laboratories and 3 companies from 8 countries will train, to the best level, a cohort of early-stage researchers using cutting-edge technologies to dissect the functions and potentials of ncRNAs through integrated multidisciplinary projects.


Grant
Agency: Cordis | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2011-ITN | Award Amount: 3.34M | Year: 2012

SPHINGONET was inspired by a common vision of its partners on the training of researchers in the field of sphingolipid homeostasis and body-wide sphingolipid signaling networks using innovative technological approaches. Dysregulation of sphingolipid balances contributes to a broad range of pathological processes, spanning neurodegeneration, asthma, autoimmune disease, insuline resistance, obesity and cancer progression. Key to defining proper sites for therapeutic intervention is a comprehensive understanding of the mechanisms of sphingolipid homeostasis and how sphingolipid-mediated signaling pathways are interconnected. In spite of its many clinical implications, progress in this field is curbed by a lack of appropriate tools to monitor, quantify and manipulate sphingolipid pools in live cells. SPHINGONETS training program is designed to close these gaps in knowledge and technology by transferring the complementary expertise of its partners to a future generation of scientists who will take a leading role in decoding the full regulatory potential of the sphingolipid signaling network and maximize its therapeutic use. By merging seven academic partners working at the forefront of sphingolipid, chemical and systems biology with three (pro)drug discovery-oriented SMEs, SPHINGONET will create a challenging interdisciplinary and clinically-relevant research environment with ample opportunities for structuring industrial projects, commercial exploitation of results, entrepreneurship and complementary education adapted to the personal needs. Thus, SPHINGONET will provide its trainees with a rounded education that, besides enhancing their career perspectives, will enable them to choose a career path in Europes academia or industry, and be successful at it while retaining ties between both these bodies.


Hannus M.,University of Regensburg | Hannus M.,Intana Bioscience GmbH | Hannus M.,SiTools Biotech GmbH | Beitzinger M.,University of Regensburg | And 6 more authors.
Nucleic Acids Research | Year: 2014

Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools - referred to as siPools - containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.


Schraivogel D.,University of Regensburg | Schindler S.G.,University of Lübeck | Danner J.,University of Regensburg | Kremmer E.,Helmholtz Center Munich | And 4 more authors.
Nucleic Acids Research | Year: 2015

MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago-TNRC6 levels. © 2015 The Author(s) .


Jakob L.,University of Regensburg | Treiber T.,University of Regensburg | Treiber N.,University of Regensburg | Gust A.,University of Regensburg | And 8 more authors.
RNA | Year: 2016

In the microRNA (miRNA) pathway, Dicer processes precursors to mature miRNAs. For efficient processing, double-stranded RNA-binding proteins support Dicer proteins. In flies, Loquacious (Loqs) interacts with Dicer1 (dmDcr1) to facilitate miRNA processing. Here, we have solved the structure of the third double-stranded RNA-binding domain (dsRBD) of Loqs and define specific structural elements that interact with dmDcr1. In addition, we show that the linker preceding dsRBD3 contributes significantly to dmDcr1 binding. Furthermore, our structural work demonstrates that the third dsRBD of Loqs forms homodimers. Mutations in the dimerization interface abrogate dmDcr1 interaction. Loqs, however, binds to dmDcr1 as a monomer using the identified dimerization surface, which suggests that Loqs might form dimers under conditions where dmDcr1 is absent or not accessible. Since critical sequence elements are conserved, we suggest that dimerization might be a general feature of dsRBD proteins in gene silencing. © 2016 Jakob et al.


Hannus S.,Intana Bioscience GmbH | Brock R.,Radboud University Nijmegen
BioSpektrum | Year: 2012

Fluorescence correlation spectroscopy (FCS) derives information on molecular interactions and concentrations from the analysis of timedependent fluctuations of a fluorescence signal, caused by diffusion of molecules through a confocal detection volume. Here, we present applications of FCS in drug discovery and molecular pharmacology, including the screening of drug-target interactions and peptide stability in crude cellular lysates, providing a physiological environment. © Springer-Verlag 2012.


Glauner H.,Radboud University Nijmegen | Ruttekolk I.R.,Radboud University Nijmegen | Hansen K.,Intana Bioscience GmbH | Steemers B.,Radboud University Nijmegen | And 4 more authors.
British Journal of Pharmacology | Year: 2010

Background and purpose: In vitro assays that determine activities of drug candidates with isolated targets have only limited predictive value for activities in cellular assays. Poor membrane permeability and off-target binding are major reasons for such discrepancies. However, it still difficult to directly analyse off-target binding at the same time as target binding, on a subcellular level. Here, we present a combination of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) as a solution to this problem. Experimental approach: The well-established dihydrofolate reductase inhibitor methotrexate and the kinase inhibitors PD173956 and purvalanol B were conjugated via polyethylene glycol linkers with the fluorophore Cy5. The cellular uptake and subcellular distribution of these compounds in single human cancer-derived cells were investigated by confocal laser scanning microscopy. In addition, molecular interactions inside the cell with the respective target proteins and off-target binding were detected simultaneously in the nanomolar range by FCCS and FCS, respectively, using cells expressing green fluorescent protein fusion proteins of dihydrofolate reductase and Abelson kinase 1. Key results: Large differences in the interaction patterns were found for these compounds. For methotrexate-Cy5, drug-target interactions could be detected and dissociation constants determined. In contrast, PD173956-Cy5 showed strong interactions with intracellular high-molecular weight structures, other than its target. Conclusions and implications: The combination of FCS and FCCS provides a powerful means to assess subcellular pharmacokinetics and dynamics of drug candidates at nanomolar concentrations. © 2010 The British Pharmacological Society.


Patent
Intana Bioscience GmbH | Date: 2013-04-25

The present invention relates to a method for producing pools of siRNA molecules suitable for RNA interference.


The present invention relates to the determination of interaction parameters of at least two analytes in cellular lysates, wherein at least one competitive agent is optionally further present.


The present invention relates to the determination of interaction parameters of at least two analytes in cellular lysates, wherein at least one competitive agent is optionally further present.

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