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.

Figure 1

Very few 5hmC-modified iMs contribute to the overall density of iMs around the TSS. (a) Overall density of iM-forming genes relative to the TSS. (b) Density of iM-forming genes having 5hmCs colocalized within 100 bp of an iM.

Figure 2

Relationship between iM potential and 5hmC density. (a) Contour plot for the sequences upstream of the TSS shows that 5hmC enrichment is associated with genes having low-iM-forming potential. (b) Contour plot for the downstream sequences shows that the 5hmC enrichment is associated with genes having high-iM-forming potentials.

Figure 3

pH denaturation of (a) C6T, (b) 5mC-C6T, and (c) 5hmC-C6T. pK_a increases slightly with 5mC modification and is lowered with 5hmC modification. The pH melting curve (d) shows substantial co-operativity when the iM contains 5hmC modification.

Figure 4

pH scans for C6T in the presence of PEG-300. (a) Melting scans of iM structure, under molecular crowding conditions, with increasing pH. (b) Fits showing that the pK_a of C6T in the presence (black) of molecular crowding agents shifts toward neutral, whereas the pK_a in the absence (blue) of crowding is in the acidic range. The other smaller cosolutes did not show any pK_a shifts.

Figure 5

CD melting trends for C6T (□), 5hmC-C6T (●), and 5mC-C6T (▲). 5hmC modification thermally destabilized the iM structure.

Figure 6

Thermal melting profiles for C6T in 10 (◊), 20 (●), 30 (▲), and 40% (■) cosolvents. Melting temperature increases with the addition of (a) dimethoxyethane, (b) 2-methoxyethanol, and (d) PEG-300. Melting temperature decreases with the addition of (c) glycerol.

Figure 7

Changes in K_obs with respect to changing water activity (a_w) for C6T at 37 °C in pH 5.4 solutions. Nearly identical results were obtained for 5hmC-C6T and 5mC-C6T, indicating that the solvent effects are indifferent to epigenetic modification.