Iminothioindoxyl Donors with Exceptionally High Cross Section for Protein Vibrational Energy Transfer.

Various protein functions are related to vibrational energy transfer (VET) as an important mechanism. The underlying transfer pathways can be experimentally followed by ultrafast Vis-pump/IR-probe spectroscopy with a donor-sensor pair of non-canonical amino acids (ncAAs) incorporated in a protein. However, so far only one donor ncAA, azulenylalanine (AzAla), exists, which suffers from a comparably low Vis extinction coefficient. Here, we introduce two novel donor ncAAs based on an iminothioindoxyl (ITI) chromophore. The dimethylamino-ITI (DMA-ITI) and julolidine-ITI (J-ITI) moieties overcome the limitation of AzAla with a 50 times higher Vis extinction coefficient. While ITI moieties are known for ultrafast photoswitching, DMA-ITI and J-ITI exclusively form a hot ground state on the sub-ps timescale instead, which is essential for their usage as vibrational energy donor. In VET measurements of donor-sensor dipeptides we investigate the performance of the new donors. We observe 20 times larger signals compared to the established AzAla donor, which opens unprecedented possibilities for the study of VET in proteins.

Introducing LNAzo: More Rigidity for Improved Photocontrol of Oligonucleotide Hybridization.

Oligonucleotide-based therapeutics have made rapid progress in clinical treatment of a variety of disease indications. Since most therapeutic oligonucleotides serve more than just one function and tend to have a prolonged lifetime, spatio-temporal control of these functions would be desirable. Photoswitches like azobenzene have proven themselves as useful tools in this matter. Upon irradiation, the photoisomerization of the azobenzene moiety causes destabilization in adjacent base pairs, leading to a decreased hybridization affinity. Since the way the azobenzene is incorporated in the oligonucleotide is of utmost importance, we synthesized locked azobenzene C-nucleosides and compared their photocontrol capabilities to established azobenzene C-nucleosides in oligonucleotide test-sequences by means of fluorescence-, UV/Vis-, and CD-spectroscopy.

Light Regulation of DNA Minicircle Dimerization by Utilizing Azobenzene C-Nucleosides.

Although DNA has the ability to form almost every desired shape, the usability of DNA nanostructures can be limited due to the lack of functionality. To functionalize nanoscale structures, light-responsive moieties like photoswitchable azobenzenes can be introduced into DNA. Upon UV irradiation, the isomerization of the azobenzene moiety causes destabilization of the neighboring base pairs leading to decreased binding ability. The linker strategy of the azobenzene to the DNA alters the performance of the switching behavior significantly. We hereby report the utilization of four different azobenzene C-nucleosides and compare their features in a nanoarchitecture model with the help of gel-electrophoresis and atomic force microscope-imaging.

Photo-Tethers for the (Multi-)Cyclic, Conformational Caging of Long Oligonucleotides.

Intramolecular circularization of DNA oligonucleotides was accomplished by incorporation of alkyne-modified photolabile nucleosides into DNA sequences, followed by a CuI -catalyzed alkyne-azide cycloaddition with bis-azido linker molecules. We determined a range of ring sizes, in which the caged circular oligonucleotides exhibit superior duplex destabilizing properties. Specific binding of a full-length 90 nt C10 aptamer recognizing human Burkitt's lymphoma cells was then temporarily inhibited by locking the aptamer in a bicircularized structure. Irradiation restored the native aptamer conformation resulting in efficient cell binding and uptake. The photo-tether strategy presented here provides a robust and versatile tool for the light-activation of longer functional oligonucleotides, noteworthy without prior knowledge on the structure and the importance of specific nucleotides within a DNA aptamer.