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We have created a hybrid i-motif composed of two DNA and two peptide nucleic acid (PNA) strands from an equimolar mixture of a C-rich DNA and analogous PNA sequence. Nano-electrospray ionization mass spectrometry confirmed the formation of a tetrameric species, composed of PNA-DNA heteroduplexes. Thermal denaturation and CD experiments revealed that the structure was held together by C-H+-C base pairs. High resolution NMR spectroscopy confirmed that PNA and DNA form a unique complex comprising five C-H+-C base pairs per heteroduplex. The imino protons are protected from D2O exchange suggesting intercalation of the heteroduplexes as seen in DNA4 i-motifs. FRET established the relative DNA and PNA strand polarities in the hybrid. The DNA strands were arranged antiparallel with respect to one another. The same topology was observed for PNA strands. Fluorescence quenching revealed that both PNA-DNA parallel heteroduplexes are intercalated, such that both DNA strands occupy one of the narrow grooves. H1'-H1' NOEs show that both heteroduplexes are fully intercalated and that both DNA strands are disposed towards a narrow groove, invoking sugar-sugar interactions as seen in DNA4 i-motifs. The hybrid i-motif shows enhanced thermal stability, intermediate pH dependence and forms at relatively low concentrations making it an ideal nanoscale structural element for pH-based molecular switches. It also serves as a good model system to assess the contribution of sugar-sugar contacts in i-motif tetramerization.

The PNA–DNA hybrid I-motif: implications for sugar–sugar contacts in i-motif tetramerization