2.1: Multiple layers of selective photolysis of caging groups

The task of this PhD student is to explore possibilities for the sequential or even orthogonal addressing of individual photolabile groups. He or she can build on recent studies in the group of Heckel which have shown for the first time that it is indeed possible to realize four layers of sequential uncaging of DNA. However neither the irradiation time nor the selectivity was optimal yet. Therefore, in a close collaboration with the predictions from the PhD thesis A1.2 existing caging group platforms will be optimized. In a close collaboration with PhD thesis A3.2 the performance of these new caging groups will be studied with (ultrafast) optical spectroscopy. The aim in this RTG is to establish a feedback loop where synthesis is supported by calculations and the resulting spectroscopic evidence can help fine tune the prediction algorithms. This PhD student will test the performance of the newly developed caging groups on simple substrates like glutamate and on DNA and RNA oligonucleotides – also in collaboration with PhD thesis B1.2.  A detailed knowledge of the photo­physical properties will allow optimization of as many layers of selective uncaging as possible.

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A2.2: Two-photon uncaging of oligonucleotides and peptides

Three-dimensional two-photon uncaging of oligonucleotides is a technique that is yet to be developed fully. The challenges arise first of all in the technical realization of two-photon uncaging of arbitrary areas in a microscope. In this respect all the required devices are functionally assembled in the group of Heckel. The second major challenge is the development of two-photon caging groups. The caging group with the highest action cross-section δ is the EANBP group developed by Goeldner and Specht. However, preliminary experiments have shown that its performance as caging group in nucleic acids is not as good. The third challenge is to find suitable assays to perform 3D two-photon uncaging with oligonucleotides and to quantify the two-photon action cross sections. This is difficult because of the extreme localization of the effect. This PhD student will therefore:

  • design optimized two-photon caging groups in collaboration with PhD thesis A1.2.
  • synthesize the caging groups with predicted superior properties.
  • explore wavelength-selective two-photon uncaging.
  • apply these caging groups to caged DNA, RNA and in collaboration with PhD thesis B4.3 to peptides.
  • develop assays for the visualization of 3D two-photon uncaging of DNA and RNA
  • develop assays for the determination of the two-photon action cross section δ.

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A2.3: Caged fluorophores for super-resolution microscopy and new strategies for the caging of DNA and RNA

This PhD student will closely work together with PhD thesis B5.1. The aim is here to provide new possibilities for super-resolution microscopy. To this aim he or she will explore the possibilities to provide fluorophores which are at first caged and hence non-fluorescent but become fluorescent upon uncaging. The better the amplitude of this effect the better the result in the microscopy application.

As second aim this PhD student will explore new ways to destabilize DNA- and RNA duplexes in a close collaboration with PhD thesis B1.3. We will conduct a structure-based approach. In preliminary experiments the Schwalbe and the Heckel groups have investigated the influence of single nucleobase-caged DNA residues both on a global and especially on a local base pair level. They have obtained data on single base pair stabilities with NMR proton exchange experiments and have also obtained NOE-based structural models. Therefore, it has become possible for the first time to really study the effects of configuration and/or steric aspects on the destabilizing capacity of caging groups in DNA. Based on this preliminary work this PhD student – together with PhD thesis B1.3 – will obtain more structures and explore and improve strategies for caging of DNA and RNA. The goal is to maximize the influence of the cage on the structure so that already one cage has a maximal destabilizing effect.

Furthermore, this PhD student will closely work together with PhD theses B1.2 and B2.1 and prepare nucleobase-caged RNA for the investigation of RNA folding pathways.

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