Tuning i-motif folding and stability with acyclic phosphonate-linked (ZNA) cytidine residues

i-Motifs (iMs) are non-canonical four-stranded DNA structures stabilized by hemi-protonated (C:H⁺C) cytosine base pairs. Under non-acidic pH conditions, cytosine deprotonation disrupts these interactions, resulting in structural unfolding. To broaden the environmental conditions that support iM formation for potential biomedical applications, we investigated the effect of substituting 2'-deoxycytidine residues with either the (<i>R</i>) or (<i>S</i>) isomers of an acyclic phosphonate-linked nucleotide (ZNA) bearing a cytosine (C) or 5-fluorocytosine (5F-C) nucleobase within the 22-mer vertebrate telomeric iM. UV melting and NMR spectroscopy analyses revealed that the presence of ZNA-C in the core of the iM decreased its stability, while 5F-C altered the iM stability across different pH ranges. Molecular dynamics (MD) provided a first glimpse of the quasi-symmetrical loops 1 and 3 in the iM formed by the native vertebrate telomeric sequence. The changes in the dynamics of the loop region in the modified sequences could be correlated with their decreased stability, despite stable base pairing occurring for both (<i>R</i>) and (<i>S</i>)-ZNA modifications.