DNA i-Motifs With Guanidino-i-Clamp Residues: The Counterplay Between Kinetics and Thermodynamics and Implications for the Design of pH Sensors

Abstract I-motif structures, adopted by cytosine-rich DNA strands, have attracted considerable interest as possible regulatory elements in genomes. Applied science exploits the advantages of i -motif stabilization under acidic conditions: i -motif-based pH sensors and other biocompatible nanodevices are being developed. Two key characteristics of i -motifs as core elements of nanodevices, i.e. , their stability under physiological conditions and folding/unfolding rates, still need to be improved. We have previously reported a phenoxazine derivative ( i -clamp) that enhances the thermal stability of the i -motif and shifts the pH transition point closer to physiological values. Here, we performed i -clamp guanidinylation to further explore the prospects of clamp-like modifications in i -motif fine-tuning. Based on molecular modeling data, we concluded that clamp guanidinylation facilitated interstrand interactions in an i- motif core and ultimately stabilized the i -motif structure. We tested the effects of guanidino- i- clamp insertions on the thermal stabilities of genomic and model i- motifs. We also investigated the folding/unfolding kinetics of native and modified i -motifs under moderate, physiologically relevant pH alterations. We demonstrated fast folding/unfolding of native genomic and model i -motifs in response to pH stimuli. This finding supports the concept of i -motifs as possible genomic regulatory elements and encourages the future design of rapid-response pH probes based on such structures. Incorporation of guanidino- i -clamp residues at/near the 5′-terminus of i -motifs dramatically decreased the apparent unfolding rates and increased the thermal stabilities of the structures. This counterplay between the effects of modifications on i -motif stability and their effects on kinetics should be taken into account in the design of pH sensors.