The effect of DNA methylation on DNA structure and DNA-small molecule interactions

The methylation of cytosines at the 5 ^th position (d5mC) is one of the most common epigenetic modifications, and alterations in methylation profile of cells are known to be involved in progression of many diseases including cancer. Increased stability of DNA accompanied by decreased flexibility upon methylation is thought to be a reason behind methylation profiles. The effects of d5mC on DNA stability and structure were investigated via systematic changes in the number and position of d5mCs in DDD. Our results revealed that d5mC substitutions changed DNA conformation and increased the stability only slightly. Next, the effect of DNA methylation on DNA-small molecule interactions was investigated using DDD and fully methylated analogue, DDD8. All the molecules examined (EtBr, Dox, Net and Hoe) had slightly higher affinity to DDD8 compared to DDD. Conversely, their effect, especially Dox’s, on DDD structure was more pronounced. Further investigations via MD simulations revealed high selectivity of Dox towards a single intercalation site where the methoxy group of Dox interacts with the methyl group of d5mC and that of two precedent dT to create a highly stable hydrophobic cluster. Hydrophobic cluster formation was not observed upon Dox binding to DDD. Our results rationalize the increased stability of DDD8 over DDD, and open new routes for the design of drugs targeting epigenetic modifications. We suggest, the design of drugs that can engage in hydrophobic interactions with methyl groups in the major groove of a 5’-dTdTd5mCdG-3’ sequence might lead the way in specific targeting of hypermethylated regions in cancer cells.