4n–1 Is a “Sweet Spot” in DNA i-Motif Folding of 2′-Deoxycytidine Homopolymers

Strands of DNA with four or more contiguous runs of 2′-deoxycytidine (dC) nucleotides have the potential to adopt i-motif folds, generally under mildly acidic conditions. Analysis of dC homo-oligonucleotide strands ranging in length from 10 to 30 nucleotides by five different pH-dependent methods identified a pattern in strand length vs stability. Beginning with dC11, which does not fold, the transition pH (pHT) increased with chain length with the addition of up to four nucleotides, after which the stability dramatically decreased, and the trend repeated this cycle up to dC27. The analysis found dCn strands of length 15, 19, 23, and 27 nucleotides (i.e., 4n–1) to have pHT values >7.2 and thermal stabilities >37 °C at pH 7.0. Model studies using thymidine nucleotides to lock in i-motif loop lengths support the conclusion that the most stable dCn i-motifs possess one nucleotide in each of the three loops and a core built of an even number of base pairs. The pattern identified from the model studies occurs with a frequency of four nucleotides at lengths of 15, 19, 23, and 27 in accordance with the results obtained for the dCn strands. This observation led us to interrogate the human genome for dCn runs. Inspection of the human genome indicates that dCn runs are enriched in critical regions of the genome (promoters, UTRs, and introns), while being depleted in coding and intergenic regions, and these findings may have biological implications. Lastly, the ability to tune i-motif stabilities by the length of the strand might be harnessed for stimulus-responsive applications in DNA scaffolds, sensors, nanotechnology, and other chemical applications.