Composite 5-methylations of cytosines modulate i-motif stability in a sequence-specific manner: Implications for DNA nanotechnology and epigenetic regulation of plant telomeric DNA

Background: The i-motif is a tetrameric DNA structure based on the formation of hemiprotonated cytosinecytosine (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, C-5m(+).C and C-5m(+).C-5m 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 CMP5 in the i-motif stem on the i-motif stability and pK(a). Results: CMP5 have strong influence on i-motif stability and pK(a) and influence these parameters in sequencespecific manner. In contrast to a general belief, i) CMP5 do not invariably stabilize the i-motif, and ii) when the CMP5 do stabilize the i-motif, the extent of the stabilization depends (in a complex manner) on the number and pattern of symmetric C-5(m)+.C-5(m) or asymmetric C-5(m)+.C base pairs in the i-motif stem. Conclusions: CMP5 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.