Minor groove tetrads: a potent and versatile capping interaction for i-motif structures

Abstract Minor groove tetrads (MGTs) have emerged as powerful structural elements capable of enhancing the stability and versatility of i-motif DNA structures. These non-canonical tetrads, formed by the minor groove association of two Watson–Crick or mismatched base pairs, act as capping platforms that reinforce hemiprotonated C:C⁺ stacks, enabling i-motif folding even at neutral pH. The resulting MGT-containing i-motifs (MGTiMs) display exceptional thermal and pH stability, tunable topology, and remarkable structural plasticity. Recent studies have revealed that MGTiMs can form compact architectures with only two C:C⁺ pairs, undergo reversible pH-dependent conformational transitions, and integrate seamlessly into duplex junctions without distorting B-DNA geometry. These insights may add new principles for rational i-motif engineering, guiding the design of predictable, homogeneous, and responsive DNA nanostructures. Furthermore, the synergy between MGT stabilization and chemical modifications, such as 2′-fluoro substitutions or fluorescent cytosine analogues, offers powerful tools for real-time structural monitoring and in-cell imaging. Beyond fundamental structural biology, MGTiMs hold strong potential for applications in biosensing, nanotechnology, and synthetic biology, providing programmable molecular systems that combine biocompatibility, robustness, and responsiveness to physiological stimuli.