Projects with a focus on RNA, aptamers, riboswitches

  • project1Characterizing structural and energetic aspects of aptamer-metabolite complexes by mass spectrometry will present several challenges: (1) metabolites are small molecules bound to the aptamer by only a few non-covalent interactions, and we will need to preserve these complexes and quantify them, in biologically relevant conditions. (2) Moreover the function is linked to conformational rearrangements, and measuring the mass alone will not suffice, therefore we will ion mobility spectrometry to reveal these conformational changes. The newly developed methods can help screening RNA libraries with regard to ligand binding, or to screen small molecule libraries with regard to binding to a specific RNA sensor.

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  • project2We have recently described aptamer-based biosensors taking advantage of loop-loop (kissing) interactions (Durand et al., Angewandte Chemie, 2014). We will make use of this technology for tayloring sensors able to respond to the presence of tumor markers in live cells, signaling their presence by a signal (fluorescence, …) The project will require the use of SELEX and biophysical methods (SPR, fluorescence anisotropy, …) for characterizing the sensor-ligand complexes.

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  • This project is about selecting, characterizing and applying RNA aptamers that recognize so-called flux-signalling metabolites, e.g. Fructose 1,6 diphosphate. These aptamers will be used in collaboration with other members of the MetaRNA network to develop molecular sensors and to monitor flux-signaling in cells and in real time. For this work, we will use a number of different techniques, such as robotics-based selection, Next Generation Sequencing (NGS), sophisticated bioinformatics, interaction analysis and others.

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  • project-3-4We recently employed RNA-based fluorescent molecules to generate allosteric assemblies, built from individual RNA domains. Within this project, novel RNA-based sensors for so-called flux-signaling metabolites will be generated and validated in vitro and in cells. We would like to unravel the dynamics and responsiveness of two conjoined RNA domains and their usefulness to monitor flux-signaling in real time. For this work, we will use a series of elaborated techniques, e.g. in vitro selection, fluorescent microscopy, FACS, and many more.

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  • image descriptionRNA-based sensors are key elements in the MetaRNA network. This project deals with the identification and characterization of RNA aptamers that can be used as flux-sensing molecules. In vitro selected metabolite-binding aptamers will be characterized by structural probing both in vitro and under cellular conditions. For this work, we will use a number of biochemical and biophysical methods to characterize RNA aptamer structures in detail.

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  • image descriptionCellular conditions often influence the structure and consequently the function of RNA molecules. Therefore, appropriate screening systems are important to identify RNA aptamers with riboswitch function. In this project, we will develop screening systems for different organisms to optimize RNA-based flux sensors. Different techniques, such as molecular cloning, microscopy and fluorescence based cell sorting (FACS), will be applied.

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  • We have recently developed the first methodology to automatically design RNA circuits (Rodrigo et al. PNAS 2012). We experimentally validated with a sirboregulator system, but we have recently expanded it to the incorporation of RNA-only cascades, RNA heterodimers, RNA-based two-component signal transduction and RNA-based transistors, among others. For this work, we will use a number of different techniques, such as phage-assisted directed evolution, computational design, microscopy, microfluidics, and many more. We aim to design RNA-based sensors by computational design and directed evolution, which will be characterised in living cells at the single-cell and population levels.

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  • We have deployed synthetic selection systems based on biosensors to couple cellular survival to production of a particular compound. Such systems are very useful for metabolic engineering projects in order to select compound producing cells from complex and diverse libraries. In order to apply this cellular technology to the optimization of biological cell factories it is necessary to develop general approaches by which the sensitivity of such synthetic networks can be tuned to gradually increase the production requirements. Using such systems we can continue to select for higher producing cells and thereby accelerate the development of cell factories.

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  • project-13Efficient cellular and in vivo delivery of RNA oligonucleotides is a major challenge, requiring either chemical modification, conjugation to or complexation with appropriate delivery ligands or nanocarriers. In the project we shall design and synthesize novel agents for in vivo intracellular delivery of RNA, and study the cellular and in vivo activity of these as well as the in vivo biodistribution of the RNA. The work shall involve organic chemical synthesis and characterization as well as some biological testing, including in vivo fluorescence imaging.

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