With respect to the biological context the research program of the RTG is divided into threeplus-one research areas All four areas are strongly interconnected with eachother as pointed out in the short project descriptions below. Each project will besupervised by several project leaders and can involve one or more PhD students.
Project area A: Imaging of transport and delivery into cells
A1: Synthesis of multifunctional transport moieties for cellular uptake in an organelleor organ-specific manner
In this project the aim is to utilize peptoidic compounds, polyamine structures or related molecules to achieve cell or organ specific uptake and to study the mechanism of cell penetration in dependence of different functionalization patterns.
A2: Biofunctional fluorogenic single chain polymer nanoparticles for intracellular transport imaging
Provision of a widely applicable tool box for the design of inherently fluorescent nano-carriers functionalized with biotargeting entities that can be finely adjusted to a wide range of celldelivery and imaging applications.
A3: Imaging of siRNA delivery and processing using wavelength-shifting siRNA probes
Development of a fluorescent siRNA probes with a sensitive and reliable readout that do not significantly interfere with the silencing efficacy and that are able to discriminate between single and double stranded condition by their emission color using a single excitation wavelength. Using these siRNA probes both intracellular transfection systems and nuclease resistance/degradation can bemonitored and compared by fluorescent imaging of siRNA uptake and processing inside the cell. This includes newly designed transport systems within this RTG (projects A1 and A2).
Project area B: Tracing of molecules inside cells
B1: Photoswitchable fluorophores for real-time imaging of bioactivity modulation
Development of a small-molecule reporter for real-time fluorescent imaging of azophotoswitch photoisomerization. This reporter will be then covalently bound to biopolymers to correlate the degree of photoisomerization with bioactivitychanges in vitro
and in vivo
. The excitation of the fluorophore and the photoisomerization wavelength should be well-separated and both preferentially higher than 450 nm to avoidcytotoxic effects of UV light during in vivo
B2: Reporter systems for live-cell FISH of bacterial communities
Development of in vivo
reporter systems on the basis of cationic lipids and cell penetrating carriers, to deliver DNA molecular beacons into bacteria for hybridization-baseddetection of specific 16S-rRNA sequences.
B3: Imaging of small molecules as cellular components by wavelength-shifting RNA aptamer probes
Development of wavelength-shifting RNA aptamer probes that are purely based on RNA together with a combination of two different dyes.
Project area C: Functional Imaging in living zebrafish and otheranimal models
C1: Nanoparticle reporters for imaging metabolites during zebrafish development
Development of a novel platform of injectable sensors for small-molecule detection, which are based on supramolecular nanoparticle-constructs bearing genetically engineered metabolite-binding proteins. The proteins are encapsulated in dye-encoded silica-nanoparticles to be shielded from interferences with endogenous macromolecular components and to increase detection quality through ratiometric measurementes and/or FRET mechanisms. These probes should enable real-time imaging of metabolite binding, and thus quantitative mapping of small-molecule analyte concentrations, such as of glucose.
C2: In vivo imaging of tissue targeted fluorescent probes to investigate adult neurogenesis and CNS regeneration
Using zebrafish as efficient screening model with the aim to develop CPPos forsystematic gene function studies in neurogenesis of the adult brain. Those transporters that cross the blood brain barrier will have a wider application and will be studied in-depth in mice to assess their potential therapeutic application. Besides the development of an imaging method for adult zebrafish we will further test other fluorophores.
Project area M: supports the RTG by development of methodologies inmicroscopy and physical chemistry
M1: Advanced fluorescence microscopy for the characterization of molecular probes
Employment of a range of advanced fluorescence microscopy techniques with single-molecule sensitivity to the photophysical and photochemical characterization of the novel probes that are being developed in the RTG, which is a clear prerequisite for their application in living systems. By using a confocal microscope with pulsed laser excitation and time-resolved fluorescence detection in up to four channels, we will study fluorescent probes freely diffusing in solution. For analyzing the data recorded in these measurements, a wide range of specialized tools are available. Moreover, we will measure the emission of the fluorescent probes, which are immobilized sparsely on suitably prepared solid supports and analyze the emission time traces. With these powerful techniques, key parameters of the fluorophores will be determined in vitro.
We also plan to employ our advanced imaging modalities for the study of the fluorescent probes developed here in their interactions with living cells and entire organisms.
M2: Investigations of ground and excited state properties of fluorescent probes in liquids by means of femtosecond spectroscopy and QM/MM methods
The primary goal is to focus on RTG-specific issues of elementary photophysical and photochemical processes of fluorescent probes using both transient absorption spectroscopy and theoretical calculations. Experimentally, femtosecond pump-probe absorption spectroscopy in solution will be applied. For a more quantitative description, these studies are combined withtheoretical calculations to assign electronic levels and elucidate intra- and intermoleculartransfer processes and their corresponding time scales. High level quantum chemical methods (MRCI, CASPT2), Molecular Dynamics (MD) simulations and combined Quantum Mechanics Molecular Mechanics (QM/MM) approaches will be used to investigate excitedstate properties and dynamics of the new compounds.