I-motifs are largely underexplored tetraplex nucleic acid structures which have been suggested to play essential biological functions and might constitute future therapeutic targets. I-motifs generally require acidic conditions to fold in vitro, a particularity which significantly complicates the use of native i-motif forming sequences for interactions studies with potential ligands and biological components (e.g. proteins). In this context, we report herein on the assembly of a peptide-DNA conjugate capable to fold at room temperature into a stable i-motif structure at neutral pH. To achieve the controlled assembly of the i-motif forming conjugate, we developed a new synthetic pathway of four successive orthogonal ligation reactions between bifunctional Crich DNA strands and a tetrafunctional cyclopeptide scaffold.