Probing i-motifs in a cellular model using a responsive nucleoside probe

Abstract

G-rich sequences and C- rich sequences which can form G-quadruplexes and imotifs have been considered as important regulatory element in oncogenes. Although G-quadruplexes have been studied extensively inside cellular condition but i-motif has been not explored. Given that C-rich sequences coexist along with gquadruplexes development of biophysical platform will be important. Here we have utilized a responsive nucleoside probe to investigate the structure of i-motif forming DNA oligonucleotides, H-telo (Human telomeric) and bcl2 (B-cell lymphoma), in aqueous buffer and inside a well-known cellular model, reverse micelles. The probe is composed of a microenvironment-sensitive fluorophore and 19F NMR isotope label, which is synthesized by coupling fluorobenzofuran with 5-iodo-2′-deoxyuridine. The probe faithfully reports the changes in the microenvironment of the water core inside reverse micelles (RM). Probe incorporated into the loop region of different Crich oligonucleotides H-telo and bcl2 does not hamper the formation of i-motif structure and its structural stability. Steady-state fluorescence and CD of modified oligonucleotides confirmed the formation of i-motif structure by these sequences at lower pH both in aqueous buffer and RM. Transition pH for the conversion of random coil to i-motif was determined by fluorescence and CD measurements. Further, 19F and 1H NMR of modified oligonucleotides reported formation of i-motif and transition from random coil to i-motif. Overall, the combination of responsive nucleoside probe and RM can be used to understand i-motif structures formed by individual C-rich sequences in near cellular environment.