An Adenine-Based Molecular Rotor as a Fluorescent Nucleobase with High Brightness

Abstract

Chemically modified nucleic acids have become a powerful platform for basic research and applied technologies. Universal nucleobases are used in PCR, sequencing, and the design of nanodevices and aptamers. Fluorescent universal nucleobases have an even wider range of applications, including the development of nucleic acid-based sensors, switches, and relay logic gates. However, few such nucleobases have been proposed to date, with most having suboptimal optical properties such as low brightness and fluorescence quenching by flanking nucleobases. Here, we propose an adenine-based molecular rotor, 7,8-dihydro-8-oxo-6-(3-methylbenzo[d]thiazol-2(3H)-ylidene)adenine (oxo-AdeBZT), as a new, remarkably bright and potent fluorescent nucleobase. Its brightness in both oligodeoxyribonucleotides (ODNs) and DNA duplexes (4200 - 10000 M-1 × cm-1) originates from a high molar extinction coefficient (averaged ε368 37000 M-1 × cm-1), provided by the appended 3-methylbenzo[d]thiazolyl moiety, and a relatively high quantum yield (0.11 – 0.27). Melting temperature and Gibbs free energy variations observed upon the incorporation of oxo-AdeBZT opposite various native nucleobases in a duplex context typically did not exceed 5 °C and 0.6 kcal/mol, respectively. The basis of these uniform hybridizing properties was unveiled using computational methods. According to molecular dynamics simulations, oxo-AdeBZT pushes the opposite nucleobase out of the DNA double helix and forms multiple hydrophobic contacts with the flanking base pairs. At the same time, the rotational mobility of the bonds between the oxo-AdeBZT-constituting heterobicycles decreases, and oxo-AdeBZT adopts a planar conformation in both ODNs and their duplexes, resulting in the light-up effect. These properties make oxo-AdeBZT a promising molecular tool for analytical, biophysical and biochemical studies.