Abstract
Genomic locations such as promoter, exon, intron, telomeric and non-telomeric regions are rich in GC-rich sequences with the potential to form G- and C-tetraplexes on both strands independently. Herein, we employed biophysical and biochemical methods to study a 34-mer C-rich DNA sequence of the myosin heavy chain β gene (MYH7β) promoter, namely HM34C for humans and the rabbit counterpart, RM34C, which differs from HM34C at three positions (three bases). Circular dichroism (CD), UV-thermal denaturation and native gel electrophoresis studies demonstrated that both the C-rich promoter segments form C-tetraplex (i-motif) structures. The CD studies revealed that HM34C forms the i-motif structure at acidic pH (5.2) in the presence of 0.1 M NaCl but remains unstructured at physiological pH. Interestingly, RM34C can form the stable i-motif structure in acidic as well as physiological pH. A shift in the positive peak from 280 nm to 275 nm with the increase in temperature from 4 °C to 30 °C was observed in temperature-dependent CD studies. UV-melting studies showed a biphasic transition for RM34C, indicating the existence of two structural species at neutral pH. In view of these findings we suggest that at physiological pH, the RM34C sequence exists in equilibrium between two structural motifs, i.e. the i-motif and homoduplex structure. This study may add to the understanding of the i-motif/homoduplex in equilibrium in physiological environments.