Abstract
i-Motifs (iMs) are quadruplex nucleic acid conformations that form in cytosine-rich regions. Because of their acidic pH dependence, iMs were thought to form only in vitro. The recent development of an iM-selective antibody, iMab, has allowed iM detection in cells, which revealed their presence at gene promoters and their cell cycle dependence. However, recent evidence emerged which appeared to suggest that iMab recognizes C-rich sequences regardless of their iM conformation. To further investigate the selectivity of iMab, we examined the binding of iMab to C-rich sequences, using a combination of pull-down and western blot assays. Here, we observe that the composition of buffers used during binding and washing steps strongly influences the selectivity of antibody binding. In addition, we demonstrate by nuclear magnetic resonance that several of the previously reported C-rich sequences, which were not expected to form iMs, actually form intermolecular iMs which are selectively recognized by iMab. Our results highlight the specificity of the iMab antibody, emphasize the importance of avoiding in vitro artifacts by optimizing DNA concentrations, blocking and washing conditions, and confirm that iMab is selective not only for intramolecular iMs but also for intermolecular iMs, while not affecting the iM conformation.