Strategies and Applications for Sequence-specific Functionalization of DNA by Methyltransferases

Epigenetics, is the study describes the (heritable) modifications in DNA structure that are not caused by changes in DNA sequence, which resultin influencing the DNA transcription. Therefore, its central role associated with certain diseases has become a focus of many researches, for example, abnormal methylations are known to be related to the developmentof many cancers, such as lung, colorectal, prostate cancer, etc. Among the vast pool of epigenetic modifications, DNA methylation is themost widely studied marker, which occurs in the form of an addition of methyl group to certain nucleotide, consequently, the transcriptional machinery gains/losses/adapts to particular actions in terms of regulatingthe target genes. Hence, developing efficient methods to monitor the changes in DNA methylation status can be with tremendous help to follow the cancer and other related diseases development, ultimately leading to the development of epigenetic diagnostic and prognostic tools. Thegoal of this PhD research aims at developing a process that enables thecapture and analysis of methylated DNA and generate a (dual colour) super-resolution methylation map by enzymatic DNA labelling and super-resolution microscopy. Enzymatic labelling utilises DNA methyltransferases ina highly specific, non-damaging way that can be used to label and capture sufficient DNA for later analysis. On the other hand, super-resolution microscopy allows precise analysis of the methylation sites at 10 to 20nm resolution level. As a proof of concept, M.MpeI and M.SssI are chosen as the methyltransferases, both of which target the CpG dinucleotide sequence for methylation. A range of oligonucleotides and plasmidor small genomic DNA molecules with known methylation sites will be subjected to analysis. The quantification of methylated and unmethylated fractions will be carried out to accurately quantify methylation levels. Human prostate cancer cell lines will be then examined in comparison to other well-known prostate cancer markers, so as to test the effectivenessof the tools. To conclude, this PhD research holds the purpose to develop tools that allow the read-out of the methylation status of DNA, and explore the potential of implementing such tools towards (cancer) diagnostic applications, both in detection diseases as well as patient classification, that may offer an outlook to prevention and cure.status: publishe