Regulation of telomeric i-motif stability by 5-methylcytosine and 5-hydroxymethylcytosine modification

Abstract

The two important epigenetic markers in the human genome, 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), are involved in gene regulation processes. As a major epigenetic target, cytosines in a C-rich DNA sequence were substituted with mC and hmC to investigate the thermal stability and pH sensitivity of the corresponding i-motifs. Circular Dichroism (CD) studies indicate the formation of i-motifs at acidic pH (<6.5) for mC- and hmC-modified DNA sequences. Thermal denaturation results suggest that DNA i-motifs are stabilized when modified with one or two mCs. However, hypermethylation with mC and single modification with hmC cause destabilization of the structure. A biomimetic crowding agent does not alter the stability effect trends resulting from mC and hmC modifications, though the corresponding i-motifs show elevated melting temperatures without significant changes in pKa values.

The effect of the neutral cytidine protonated analogue pseudoisocytidine on the stability of i-motif structures

“…Whereas psC located at the ends of the C-stack stabilizes the i-motif, substitutions in central position provoke the opposite effect. This destabilization is similar to that found for 5-hydroxymehtylcystosine 22 or UNA 33 , and not as pronounced as other destabilizing modifications like acyclic threoninol derivatives 34 .…”

“…The stabilization observed for CC9 is not as high as that observed for 2′-F-araC substitutions 25 , but comparable with the effect of 5-methylcytosines 22 , LNA 23 and other stabilizing sugar substitutions 31 . As in the case of LNA 32 , the effect of psC substitutions on the overall pH T is highly dependent on their particular position.…”

“…The possibility of increasing the stability of the i-motif at physiological conditions has attracted much attention in recent years. With this aim, a number of chemical modifications have been tested by different groups, but most of them have led to destabilization 21, 22 . The exceptions are LNA and PNA for some selected sequences 23, 24 and, especially, the 2′-F-araC substitution, which affords dramatic stabilization over a wide range of pH and nucleotidic sequences 25 .…”

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.…”