• RNA
• fluorescence
• DNA
• biosensor
• AIE
• TPE

TPE dyes have attracted increasing attention due to unique photophysical properties, also connected with aggregation-induced emission (AIE). AIE is opposite to aggregation-caused quenching (ACQ) effect, which has limited traditional fluorescent organic dyes so far. Moreover, TPE derivatives can be used as fluorescent probes in order to detect biomacromolecules. For these reasons, this project aimed to synthesize a TPE derivative, having water-soluble and light-up characteristics, and to test its possible binding towards DNA and RNA polynucleotides.
The selected dye is 1,2-bis(4-((triethylammonium)butoxy)phenyl)-1,2-tetraphenylethene dibromide (BTATPE). BTATPE was already tested with DNA (in particular oligoG-quadruplex oligonucleotides) and the protein bovine serum albumin (BSA). On the contrary, the behaviour with polynucleotides as natural and synthetic DNAs and RNAs is not yet defined.
BTATPE was synthesized by means of a McMurry coupling between two 4-(4-bromobutoxy)benzophenone molecules. The coupling product was 1,2-bis(4-(4-bromobutoxy)phenyl)-1,2-diphenylethene (DBTPE) which was converted in BTATPE by a reaction with triethylamine. All products were characterized by 1H- and 13C-NMR analysis.
The interaction between BTATPE and natural (calf thymus) DNA was studied through spectrophotometric and spectrofluorimetric tests (absorbance and fluorescence titrations, melting experiments). Besides, viscosity measurements for the BTATPE/DNA system were performed to clarify the interaction mechanism.
In order to test the possible influence of the different DNA base pairings and relevant double helix geometry towards DNA binding, poly(dG)·poly(dC) and poly(dA)·poly(dT) were also employed. Moreover, the binding affinity of BTATPE towards synthetic RNAs as single stranded poly(rA), and double poly(rA)·poly(rU) and triple poly(rA)·2poly(rU) helices was investigated.
A very different behavior is observed between natural or synthetic DNAs or RNAs. The dye does not bind to any of the single, double or triple stranded RNAs. On the other hand, BTATPE strongly binds to all DNAs, causing a dramatic stabilization of all the double helices but with effects (photophysical behavior and signal stability more than binding parameters) which strongly depend on the polynucleotide. The viscosimetric tests do not enlighten a significant DNA elongation and the enthalpy variation calculated by fluorescence titrations is negative but not so high. On the whole it can be concluded that the binding to DNA is a complex binding mode showing both intercalative and groove binding features. Groove binding is fundamental to discriminate between AT or GC base pairings and between DNAs and RNAs (and the relevant helix geometries). On the other hand, the ΔTm values of 20 °C or higher can be explained only in the presence of intercalators. Docking tests confirmed that both groove binding and dye penetration into the helix are possible, independent of the BTATPE isomeric form. The two binding mode will coexist, and their relative contribution will be a function not only of the biosubstrate but also of the dye/polymer ratio.