Incorporation of a 4-(dimethylamino)azobenzene moiety at 5'-end of TBA and T30695 aptamers: structural characterization of the resulting conjugated G-quadruplexes

The synthesis and the structural characterization of azobenzene-conjugated GQs were formed. Firstly, we synthesized the R 4-(dimethylamino)azobenzene glycerol derivative (4-DMAzo). Trans-cis photo-isomerisation of 4-DMAzo can be induced by violet light, suitable to be used on DNA systems (1). Furthermore, the two hydroxyl groups of the glycerol linker inserted on one benzene ring are used to convert the azobenzene derivative into the appropriate phosphoramidite building block, thus allowing to the synthesis of 4-DMAzo 5'-end conjugated TBA and T30695. TBA forms a chair-like monomolecular and antiparallel GQ (2), whereas parallel-stranded monomers of T30695-GQs stack at 5'-ends to form very stable GQ dimers (3). We have explored by CD, CD melting, UV fluorescence and Electrophoresis Mobility Shift Assay (EMSA) techniques, the folding properties of the two G-quadruplex forming aptamers. From the collected data it appeared that the conjugated sequences are able to fold into GQs very similar to that of the corresponding unmodified aptamers. CD melting analyses evidenced that the difference between the melting temperatures of conjugated DMAzo-TBA-GQ and TBA is∼-6°C whereas that of DMAzo-T30695-GQ and T30695 was ∼+10°C. Furthermore, cis-trans photo-isomerisation of DMAzo moiety conjugated at 5'-end GQs, was preliminarily explored by UV-Vis spectroscopy. Spectra of free DMAzo, of 5'-DMAzo-TBA and 5'-DMAzo-T30695 were performed before and after irradiation by LED light at 435 nm. We also explored the fluorescence proprieties of the two conjugated GQ sequences in comparison to that of TBA and T30695 (4). In particular, steady-state emission spectra were acquired at 25 °C using buffered phosphate solution at pH 7.0 or ethanol/water as solvents. The preliminary results show that the intrinsic fluorescence of the GQs depends on the presence of DMAzo moiety at 5'-end as well as the medium used as solvent.