Selective Recognition and Ultra-Sensitive Detection of i-Motif DNA Using a Novel Fluorescent Probe with PCA-Enhanced Quantification

<i>i</i>-Motif (iM) is a noncanonical nucleic acid secondary structure that is implicated in gene regulation and disease progression. However, the development of selective ligands and fluorescent probes for iM recognition and detection remains limited, hindering the detailed study of iM structural dynamics and their potential biomedical relevance. In this study, we introduce BT-Cy-1, a novel fluorescent probe that enables in vitro selective recognition and ultrasensitive detection of iM structures. Although BT-Cy-1 interacts with a variety of DNA conformations, its multichannel fluorescence response, when analyzed via principal component analysis, allows for reliable discrimination among iMs, G-quadruplexes, double-stranded, and single-stranded DNA, including unfolded or non-iM-forming cytosine-rich sequences. The probe further enables accurate and linear quantification of iM DNA, with a detection limit as low as 3.77 nM. To assess its performance under biologically relevant conditions, BT-Cy-1 was tested in diluted fetal bovine serum, where it retained both structural discrimination and detection sensitivity. This work provides a robust in vitro platform for the quantitative analysis of noncanonical DNA structures, serving as a foundation for future applications in nucleic acid-based diagnostics and molecular recognition technologies.