Magnesium and molecular crowding of the cosolutes stabilize the i-motif structure at physiological pH

Most of the important genomic regions, especially the G,C rich gene promoters, consist of sequences with potential to form G,C-tetraplexes on both the DNA strands. In this study, we used three C-rich oligonucleotides (11Py, 21Py, and HTPy), of which 11Py and 21Py are located at various transcriptional regulatory elements of the human genome while HTPy sequence is a C-rich strand of human telomere sequence. These C-rich oligonucleotides formed i-motif structures, verified by Circular Dichroism (CD), UV absorption melting experiments, and native gel electrophoresis. The CD spectra revealed that 11Py and 21Py form i-motif structures at acidic pH values of 4.5 and 5.7 in the presence of 100 mM NaCl but remain unstructured at pH 7.0. However, 21Py can form stable i-motif structure even at neutral pH in presence of 1 mM MgCl₂ . UV-thermal melting studies showed stabilization of 21Py i-motif at pH 5.7 in the presence of Na⁺ or K⁺ with increasing concentration of MgCl₂ or CaCl₂ from 1 to 10 mM. Significant shift in the CD peak of HTPy sequence was observed as the positive peak from 286 nm shifted to 276 nm while the negative peak from 265 to 254 nm. Further, inevitable necessity of 1 mM Mg²⁺ to form i-motif structure at neutral pH was observed. Under similar ionic conditions and neutral pH, all the three C-rich sequences were able to form stable i-motif structures (11Py, 21Py) or altered i-motif/homoduplex structures (HTPy) in the presence of MgCl₂ and cell mimicking molecular crowding conditions of 40 wt% PEG 200. It is concluded that presence of Mg²⁺ ions and molecular crowding agents induce and stabilize i-motif structures at physiological solution environment.