There are cytosine-rich regions in the genome that bind protons with high specificity. Thus protonated C-rich sequence may undergo folding to tetraplex structures called i-motifs. Therefore, one can regard such specific C-rich oligonucleotides as aptamers that recognize protons and undergo conformational transitions. Proper labeling of the aptamer with a fluorescent tag constitutes a platform to construct a pH-sensitive aptasensor. Since the hemiprotonated C-C⁺ base pairs are responsible for the folded tetraplex structure of i-motif, we decided to substitute one of cytosines in an aptamer sequence with its fluorescent analogue, 1,3-diaza-2-oxophenothiazine (tC). In this paper we report on three tC-modified fluorescent probes that contain RET related sequences as a proton recognizing aptamer. Results of the circular dichroism (CD), UV absorption melting experiments, and steady-state fluorescence measurements of these tC-modified i-motif probes are presented and discussed. The pH-induced i-motif formation by the probes resulted in fluorescence quenching of tC fluorophore. Efficiency of quenching was related to the pH variations. Suitability of the sensor for monitoring pH changes was also demonstrated.
Keywords: aptamer probe; fluorescent cytosine analogue; i-motif; pH sensing.