BACKGROUND: The i-motif is a tetrameric DNA structure based on the formation of hemiprotonated cytosine-cytosine (C+.C) base pairs. i-motifs are widely used in nanotechnology. In biological systems, i-motifs are involved in gene regulation and in control of genome integrity. In vivo, the i-motif forming sequences are subjects of epigenetic modifications, particularly 5-cytosine methylation. In plants, natively occurring methylation patterns lead to a complex network of C+.C, 5mC+.C and 5mC+.5mC base-pairs in the i-motif stem. The impact of complex methylation patterns (CMPs) on i-motif formation propensity is currently unknown. METHODS: We employed CD and UV-absorption spectroscopies, native PAGE, thermal denaturation and quantum-chemical calculations to analyse the effects of native, native-like, and non-native CMPs in the i-motif stem on the i-motif stability and pKa. RESULTS: CMPs have strong influence on i-motif stability and pKa and influence these parameters in sequence-specific manner. In contrast to a general belief, i) CMPs do not invariably stabilize the i-motif, and ii) when the CMPs do stabilize the i-motif, the extent of the stabilization depends (in a complex manner) on the number and pattern of symmetric 5mC+.5mC or asymmetric 5mC+.C base pairs in the i-motif stem. CONCLUSIONS: CMPs can be effectively used to fine-tune i-motif properties. Our data support the notion of epigenetic modifications as a plausible control mechanism of i-motif formation in vivo. GENERAL SIGNIFICANCE: Our results have implications in epigenetic regulation of telomeric DNA in plants and highlight the potential and limitations of engineered patterning of cytosine methylations on the i-motif scaffold in nanotechnological applications.

Central European Institute of Technology Masaryk University Brno 625 00 Czech Republic

Central European Institute of Technology Masaryk University Brno 625 00 Czech Republic; Department of Chemistry Faculty of Science Masaryk University Brno 625 00 Czech Republic

Institute of Biophysics of the Czech Academy of Sciences Kralovopolska 135 612 65 Brno Czech Republic

Institute of Biophysics of the Czech Academy of Sciences Kralovopolska 135 612 65 Brno Czech Republic; Central European Institute of Technology Masaryk University Brno 625 00 Czech Republic

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