Epigenetic Modification, Dehydration, and Molecular Crowding Effects on the Thermodynamics of i-Motif Structure Formation from C-Rich DNA

Abstract

DNA sequences with the potential to form secondary structures such as i-motifs (iMs) and G-quadruplexes (G4s) are abundant in the promoters of several oncogenes and, in some instances, are known to regulate gene expression. Recently, iM-forming DNA strands have also been employed as functional units in nanodevices, ranging from drug delivery systems to nanocircuitry. To understand both the mechanism of gene regulation by iMs and how to use them more efficiently in nanotechnological applications, it is essential to have a thorough knowledge of factors that govern their conformational states and stabilities. Most of the prior work to characterize the conformational dynamics of iMs have been done with iM-forming synthetic constructs like tandem (CCT)n repeats and in standard dilute buffer systems. Here, we present a systematic study on the consequences of epigenetic modifications, molecular crowding, and degree of hydration on the stabilities of an iM-forming sequence from the promoter of the c-myc gene. Our results indicate that 5-hydroxymethylation of cytosines destabilized the iMs against thermal and pH-dependent melting; contrarily, 5-methylcytosine modification stabilized the iMs. Under molecular crowding conditions (PEG-300, 40% w/v), the thermal stability of iMs increased by ∼10 °C, and the pKa was raised from 6.1 ± 0.1 to 7.0 ± 0.1. Lastly, the iM’s stability at varying degrees of hydration in 1,2-dimethoxyethane, 2-methoxyethanol, ethylene glycol, 1,3-propanediol, and glycerol cosolvents indicated that the iMs are stabilized by dehydration because of the release of water molecules when folded. Our results highlight the importance of considering the effects of epigenetic modifications, molecular crowding, and the degree of hydration on iM structural dynamics. For example, the incorporation of 5-methylycytosines and 5-hydroxymethlycytosines in iMs could be useful for fine-tuning the pH- or temperature-dependent folding/unfolding of an iM. Variations in the degree of hydration of iMs may also provide an additional control of the folded/unfolded state of iMs without having to change the pH of the surrounding matrix.

Effect of Interior Loop Length on the Thermal Stability and pKa of i-Motif DNA

“…Given our 16 , and others’, 17, 18 data on the effect of PEG on iM pK a , we investigated the difference between the pH determined by electrode measurement and the activity of protons in the PEG solutions as detected by small molecules. We examined five well-known pH indicators and cytidine monophosphate by monitoring the change in UV-Vis absorbance with change in pH in 30 mM sodium cacodylate buffer with and without 40% PEG-300.…”

Stabilization of Telomeric I‐Motif Structures by (2′S)‐2′‐Deoxy‐2′‐C‐Methylcytidine Residues

“…Some stabilization of the i‐motif at neutral pH was observed, but these results were obtained for short tetrameric structures and are difficult to extrapolate to biologically relevant intramolecular i‐motifs. As cytidine is subject to epigenetic modifications, the presence of 5‐methyl‐C and 5‐hydroxymethyl‐C in the C‐rich telomere sequence has been studied, thus showing small changes in the stability of the modified i‐motifs . Interestingly, it has been observed that the inclusion of ribocytidines destabilizes i‐motifs, but arabino‐cytidines have the opposite effect .…”

“…Base modifications such as 5‐methylcytosine and 5‐hydroxymethylcytosine are permissive bases that can be adapted to i‐motif structures. They show position‐dependent stability and different behavior under experimental crowding conditions . These bases are important epigenetic markers in the human genome.…”

Stabilization of the i-motif structure by the intra-strand cross-link formation

“…15 Nevertheless, only a few studies reported the i-motif folding at neutral pH. [16][17][18] In this study, we hypothesized that the intra-strand cross-link in the i-motif structure might increase its stability in terms of both pH-dependency and thermal denaturation. We have already reported the 4-vinyl-substituted analog of thymidine (T-vinyl) as an efficient cross-linking nucleobase for adenine and thymine in the close proximity of the target strand.…”

“…15 Nevertheless, only a few studies reported the i-motif folding at neutral pH. [16][17][18] In this study, we http://dx. …”