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.