Optimizing Chemical Functionalization Reactions to Sequence RNA 5-Formylcytosine at Single-Nucleotide Resolution

Abstract

The single site on the C-34 wobble position of methionine mitochondrial tRNA (mt-tRNA-Met) for 5-formylcytosine (f5C) is known but the modification’s distribution throughout the cell is not well understood. Preliminary quantification data from the Kleiner Lab identified the presence of f5C in polyadenylated RNA. Now, efforts to determine the distribution of f5C at single-nucleotide resolution are necessary to determine the function these newly identified sites may have. In order to do so, I developed a method to enrich and sequence f5C by optimizing chemical sequencing techniques previously studied in both DNA and RNA contexts. Examining the impact of local environment and structural motif on the percent CT conversion generated by small molecule chemical reactions with f5C provides a greater understanding of the malononitrile and pyridine borane chemical functionalization reactions used to both sequence the epitranscriptome and identify additional f5C sites. My results indicate that the identity of the bases immediately surrounding each f5C site does not seem to have major implications on the efficiency of these chemical reactions, while a more structured element causes a significant decrease in percent conversion during pyridine borane treatment. However, the percent conversion at this structured motif can be recovered after treating substrates with 2-picoline-borane, a small molecule similar to pyridine borane that reacts in denaturing conditions. Overall, the use of chemical sequencing using these three small molecules can help design a more efficient and generalizable technique to both unlock the specific locations and quantify this modification across the epitranscriptome.