High resolution crystal structure of a KRAS promoter G-quadruplex reveals a dimer with extensive poly-A π-stacking interactions for small-molecule recognition.

Aberrant KRAS signaling is a driver of many cancers and yet remains an elusive target for drug therapy. The nuclease hypersensitive element of the KRAS promoter has been reported to form secondary DNA structures called G-quadruplexes (G4s) which may play important roles in regulating KRAS expression, and has spurred interest in structural elucidation studies of the KRAS G-quadruplexes. Here, we report the first high-resolution crystal structure (1.6 Å) of a KRAS G-quadruplex as a 5'-head-to-head dimer with extensive poly-A π-stacking interactions observed across the dimer. Molecular dynamics simulations confirmed that the poly-A π-stacking interactions are also maintained in the G4 monomers. Docking and molecular dynamics simulations with two G4 ligands that display high stabilization of the KRAS G4 indicated the poly-A loop was a binding site for these ligands in addition to the 5'-G-tetrad. Given sequence and structural variability in the loop regions provide the opportunity for small-molecule targeting of specific G4s, we envisage this high-resolution crystal structure for the KRAS G-quadruplex will aid in the rational design of ligands to selectively target KRAS.

Epigenetic modification of cytosines fine tunes the stability of i-motif DNA.

i-Motifs are widely used in nanotechnology, play a part in gene regulation and have been detected in human nuclei. As these structures are composed of cytosine, they are potential sites for epigenetic modification. In addition to 5-methyl- and 5-hydroxymethylcytosine modifications, recent evidence has suggested biological roles for 5-formylcytosine and 5-carboxylcytosine. Herein the human telomeric i-motif sequence was used to examine how these four epigenetic modifications alter the thermal and pH stability of i-motifs. Changes in melting temperature and transitional pH depended on both the type of modification and its position within the i-motif forming sequence. The cytosines most sensitive to modification were next to the first and third loops within the structure. Using previously described i-motif forming sequences, we screened the MCF-7 and MCF-10A methylomes to map 5-methylcytosine and found the majority of sequences were differentially methylated in MCF7 (cancerous) and MCF10A (non-cancerous) cell lines. Furthermore, i-motif forming sequences stable at neutral pH were significantly more likely to be epigenetically modified than traditional acidic i-motif forming sequences. This work has implications not only in the epigenetic regulation of DNA, but also allows discreet tunability of i-motif stability for nanotechnological applications.