Probing nucleic acid conformations and recognition by fluorescence and 19f nmr using dual purpose nucleoside analogs

Abstract

Guanine- and cytosine-rich sequences present in the promoter region adopt non-canonical structural motifs called G-quadruplexes (GQs) and i-motifs (iMs), which have been shown to regulate gene expression at different levels. While dysfunction of these elements are implicated in cancer and other diseases, targeting them using small molecule ligands and probes remains a major challenge. These motifs show high degree of structural polymorphism with complex dynamics in vitro, and it is not clear, which topology is biologically relevant. Hence, it is important to develop robust tools that would provide information on the conformational equilibrium and topology-specific recognition properties in both cell-free and cellular environments. In this context, we developed multifunctional nucleoside probes to study nucleic acid structures and their interaction with small molecule ligands by fluorescence and 19 F NMR techniques. The modified nucleoside is composed of a trifluoromethyl-benzofuran-2-yl moiety (3FBF) at the C5 position of 2and#8242;- deoxyuridine, which functions as a responsive fluorescent and 19 F NMR probe. The nucleoside analog is highly microenvironment-sensitive and suitable for nucleic acid analysis by the above two important techniques. In the first study system, the analog was used to investigate GQ structures adopted by oncogenic EGFR promoter sequence. The probe helped in quantifying the GQ structural equilibrium as a function of change in ionic conditions. Notably, results indicate that parallel GQ topology is a predominant component under physiological conditions. Interestingly, in a cellular environment (in frog egg lysate and extract), the modified probe reveals that EGFR ON prefers to fold into a hybrid GQ topology. Next, the nucleoside analog was used in investigating the conformational equilibrium of different iM structures formed by C-rich telomeric repeat and oncogenic Braf promoter sequences. Studies indicate that iM structural equilibrium is very sensitive to changes in pH and temperature.