biostudies-literatureSkolakova PCzech Science FoundationERDF2950-2962https://www.ebi.ac.uk/biostudies/studies/S-EPMC10085675biostudies-literatureUnknown51(6)Cytosine-rich DNA regions can form four-stranded structures based on hemi-protonated C.C+ pairs, called i-motifs (iMs). Using CD, UV absorption, NMR spectroscopy, and DSC calorimetry, we show that model (CnT3)3Cn (Cn) sequences adopt iM under neutral or slightly alkaline conditions for n > 3. However, the iMs are formed with long-lasting kinetics under these conditions and melt with significant hysteresis. Sequences with n > 6 melt in two or more separate steps, indicating the presence of different iM species, the proportion of which is dependent on temperature and incubation time. At ambient temperature, kinetically favored iMs of low stability are formed, most likely consisting of short C.C+ blocks. These species act as kinetic traps and prevent the assembly of thermodynamically favored,Nucleic acids researchDNA i-motif formation at neutral pH is driven by kinetic partitioning.PMC1008567519-26041XTrantirek LSubert DMergny JLVorlickova MSkolakova PPalacky JRenciuk DGajarsky MfalseDNA i-motif formation at neutral pH is driven by kinetic partitioning.Cytosine-rich DNA regions can form four-stranded structures based on hemi-protonated C.C+ pairs, called i-motifs (iMs). Using CD, UV absorption, NMR spectroscopy, and DSC calorimetry, we show that model (CnT3)3Cn (Cn) sequences adopt iM under neutral or slightly alkaline conditions for n > 3. However, the iMs are formed with long-lasting kinetics under these conditions and melt with significant hysteresis. Sequences with n > 6 melt in two or more separate steps, indicating the presence of different iM species, the proportion of which is dependent on temperature and incubation time. At ambient temperature, kinetically favored iMs of low stability are formed, most likely consisting of short C.C+ blocks. These species act as kinetic traps and prevent the assembly of thermodynamically favored,2023-01-01T00:00:00Z2023 Apr2025-04-26T00:07:27.402Z2025-04-06T09:32:48.638ZS-EPMC100856753691210210.1093/nar/gkad119