Stabilization of VEGF i-motif structure by CpG methylation

Abstract

The intercalated motif (i-motif) is a non-canonical nucleic acid structure formed by intercalated hemi-protonated cytosine base pairs (C-C⁺) under acidic conditions. The i-motif structure formation is involved in biological processes such as transcription regulation. Therefore, the identification of factors controlling i-motif formation is important in elucidating the cellular functions it controls. We previously reported that the VEGF G-quadruplex structure is stabilized by CpG methylation. In this study, the effect of CpG methylation on the stability of the VEGF i-motif structure was investigated. The VEGF i-motif-forming oligonucleotide contains four cytosines on CpG sites, and three of the four cytosines (C4, C15, and C20) are involved in C-C⁺ formation in the i-motif structure. Circular dichroism (CD) spectra analysis demonstrated that full CpG methylation increased the pH of mid transition (pH_T) of the i-motif structure by 0.1, and the melting temperature (T_m) by 5.1 °C in 25 mM sodium cacodylate buffer at pH 5.0. Moreover, single methylation at C4, C15, and C20 increased T_m by 0.5, 1.7, and 2.0 °C in the buffer, respectively. These results demonstrated that CpG methylation stabilized the VEGF i-motif structure.

Keywords: CpG methylation; Thermal stability; VEGF; i-motif.