Unveiling the unusual i-motif-derived architecture of a DNA aptamer exhibiting high affinity for influenza A virus

Non-canonical nucleic acid structures have been shown the capacity to selectively interact with proteins, thereby exerting influence over various intracellular processes. Among these structures, both natural and artificial G-quadruplexes have been extensively studied in relation to their structure-activity relationships. In contrast, the role of i-motifs remains incompletely evaluated. In this study the artificial aptamer BV42 possessing a high affinity for hemagglutinin of influenza A virus was proven to contain the i-motif structure even at neutral pH. However, conformational heterogeneity of BV42 poses challenges for in-depth structural investigations. Using molecular dynamics simulations and introducing chemical modifications for molecular probing, a putative binding site in the aptamer was suggested. These findings have enabled us to redesign the aptamer, eliminating the conformational diversity associated with the i-motif while preserving its binding affinity. Subsequent validation through NMR spectroscopy confirms the presence of the i-motif/duplex junction with the 3-cytosine loop located near the junction. This study elucidates a distinctive instance of an unusual nucleic acid architecture involved in molecular recognition, thereby shedding light on the structural peculiarities of functional i-motifs.