Hysteresis in poly-2'-deoxycytidine i-motif folding is impacted by the method of analysis as well as loop and stem lengths.

In DNA, i-motif (iM) folds occur under slightly acidic conditions when sequences rich in 2'-deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pHT ) between the folded and unfolded states. Two different experiments to determine pHT values via circular dichroism (CD) spectroscopy were performed on poly-dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pHT values were dependent on the titration experiment performed, and (2) pH-induced denaturing or annealing processes produced isothermal hysteresis in the pHT values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly-dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly-dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pHT parameter measured, and hysteresis was observed in the pHT and Tm values.

Rapid Screen of Potential i-Motif Forming Sequences in DNA Repair Gene Promoters.

We have studied the in vitro stability of 25 potential i-motif-forming DNA sequences found within the promoter regions of 18 different human DNA repair genes. Three widely available methods of characterization were used to rapidly assess i-motif folding and stability and comprise a simple screen for preliminary identification of physiologically relevant i-motif forming sequences. Four highly pH-stable candidate sequences were identified exhibiting pH transitions (pH at which 50% of the oligodeoxynucleotides in solution are folded) at or above pH 6.6, thermal melting temperatures above 37 °C and isothermal UV difference spectra characteristic of 2'-deoxycytidine imino-nitrogen protonation. These newly identified i-motif forming sequences could represent novel targets for understanding and modulating human DNA repair gene expression.

Unusual Isothermal Hysteresis in DNA i-Motif pH Transitions: A Study of the RAD17 Promoter Sequence.

We have interrogated the isothermal folding behavior of the DNA i-motif of the human telomere, dC19, and a high-stability i-motif-forming sequence in the promoter of the human DNA repair gene RAD17 using human physiological solution and temperature conditions. We developed a circular-dichroism-spectroscopy-based pH titration method that is followed by analysis of titration curves in the derivative domain and found that the observed pH-dependent folding behavior can be significantly different and, in some cases, multiphasic, with a dependence on how rapidly i-motif folding is induced. Interestingly, the human telomere sequence exhibits unusual isothermal hysteresis in which the unfolding process always occurs at a higher pH than the folding process. For the RAD17 i-motif, rapid folding by injection into a low-pH solution results in triphasic unfolding behavior that is completely diminished when samples are slowly folded in a stepwise manner via pH titration. Chemical footprinting of the RAD17 sequence and pH titrations of dT-substituted mutants of the RAD17 sequence were used to develop a model of RAD17 folding and unfolding. These results may provide valuable information pertinent to i-motif use in sensors and materials, as well as insight into the potential biological activity of i-motif-forming sequences under stepwise or instantaneous changes in pH.

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.