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.

Emerging roles of i-motif in gene expression and disease treatment

“…Based on mechanisms of polyamine binding to i-motifs, additional research is needed to identify additional ligands with greater affinity by altering the molecular length of the polyamines. Kinetic analysis has shown that the addition of 5′-terminal guanidino-i-clamp ( Tsvetkov et al, 2019 ) or i-clamp ( Tsvetkov et al, 2018 ) can decrease the unfolding rate of synthetic i-motifs, which guarantees the stability of i-motifs. It is also possible to become unstable given the steric hindrance.…”

Ratiometric i-Motif-Based Sensor for Precise Long-Term Monitoring of pH Micro Alterations in the Nucleoplasm and Interchromatin Granules

“…Previously tested approaches to improving iM stability in neutral medium include the incorporation of non-native residues (nucleoside analogues) or extra structural elements (loops) into the iM structure and the usage of cyclopeptide scaffolds. − Also, intrinsically (semi)­stable iMs can be found among genomic sequences that contain four long cytosine tracts (C 5+ ) separated by short loops or dC homopolymers following the “4n-1” rule. ,, In contrast to bimodular (iM+hairpin) structures that typically require an additional complementary chain to induce concentration-dependent rearrangements, genomic unimolecular iMs show concentration-independent folding/unfolding. In the recent study, we examined a genomic dC homopolymer ATX labeled with terminal fluorophore and quencher residues.…”