Sequencing the Mono-Methylated Derivatives of Cytidine

Project Summary The goal of this project is to develop a prototype sequencing platform and the associated methodology capable of directly sequencing the mono-methylated derivatives of cytosine. Recently, LC-MS/MS methods to assess the presence of the mono-methylated isomers of cytosine, i.e., 5-methylcytosine (m5C), 4-methylcytidine (m4C), 3- methylcytidine (m3C), 2`-O-methylcytidine (Cm), as well as the bismethyl derivative 5-methyl-2`-O-methylcytidine (m5Cm), have made promising discoveries that these modifications change when cells are exposed to oxidative and inflammatory stress, micronutrient stress, and heat shock stress. Moreover, the placement and extent of methylated cytosine RNA nucleotides is now believed to be important in neurodevelopment, appears to impact intelligence, and changes with cancer, eczema, and metabolic disorders. However, no technology currently exists that is capable of sequencing and quantifying these chemical marks, severely limiting the field’s understanding and potential utilization of these modifications as biomarkers (for diagnostics, prognostics, and therapeutic purposes, as examples). In order to enable the proposed feat, during this project, we will specifically be developing a novel nanopore-based reader with the sensitivity to directly differentiate the mono-methylated isomers of cytosine as well as the associated sequencing platform for semi-automated, stable, high-accuracy sequencing measurements. We will then demonstrate and optimize the developed sequencing methodology through various sequencing demonstrations, including sequencing and mapping the mono-methylated derivates of cytosine across RNA from two different cell lines. Upon the completion of this project, we will have developed, optimized, and fully demonstrated the world’s first functioning prototype sequencer capable of directly sequencing the mono-methylated derivatives of cytosine.