Manuela A. H. Fichte

Three-Dimensional Control of DNA Hybridization by Orthogonal Two-Color Two-Photon Uncaging.

We successfully introduced two-photon-sensitive photolabile groups ([7-(diethylamino)coumarin-4-yl]methyl and p-dialkylaminonitrobiphenyl) into DNA strands and demonstrated their suitability for three-dimensional photorelease. To visualize the uncaging, we used a fluorescence readout based on double-strand displacement in a hydrogel and in neurons. Orthogonal two-photon uncaging of the two cages is possible, thus enabling complex scenarios of three-dimensional control of hybridization with light.

Three-Dimensional Protein Networks Assembled by Two-Photon Activation†

Spatial and temporal control over chemical and biological processes plays a key role in life and material sciences. Here we synthesized a two-photon-activatable glutathione (GSH) to trigger the interaction with glutathione S-transferase (GST) by light at superior spatiotemporal resolution. The compound shows fast and well-confined photoconversion into the bioactive GSH, which is free to interact with GST-tagged proteins. The GSH/GST interaction can be phototriggered, changing its affinity over several orders of magnitude into the nanomolar range. Multiplexed three-dimensional (3D) protein networks are simultaneously generated in situ through two-photon fs-pulsed laser-scanning excitation. The two-photon activation facilitates the three-dimensional assembly of protein structures in real time at hitherto unseen resolution in time and space, thus opening up new applications far beyond the presented examples.

A two-photon-photocleavable linker for triggering light-induced strand breaks in oligonucleotides

We synthesized a two-photon-sensitive photocleavable linker based on the 7-diethylaminocoumarin structure and introduced it successfully into DNA strands. First, we demonstrated the inducibility of strand scissions upon irradiation at 365 nm. To verify and visualize the two-photon activity, we used a fluorescence assay based on a DNA strand displacement immobilized in a hydrogel. Additionally, we investigated its use in a new class of DNA decoys that are able to catch and release nuclear factor κB (NF-κB) by using light as an external trigger signal. In cell culture we were able to show the regulation of NF-κB-controlled transcription of green fluorescent protein.