BioProject

A main limitation in assessing the location of epitranscriptomic modifications in RNA derives from a lack of methods that accurately capture location and levels. More...

A main limitation in assessing the location of epitranscriptomic modifications in RNA derives from a lack of methods that accurately capture location and levels. We previously demonstrated that the RNA modification N4-acetylcytidine (ac4C) can be mapped at base-resolution through sodium borohydride (NaBH4) reduction to tetrahydroacetylcytidine (H4-ac4C), followed by cDNA synthesis to misincorporate adenine opposite reduced ac4C sites, culminating in C:T mismatches at acetylated cytidines (RedaC:T). However, this process is generally inefficient, resulting in a maximum of about 25 percent C:T mismatches at a fully modified site in 18S rRNA. Considering that ac4C locations in other substrates including mRNA are unlikely to reach full penetrance, this method is not ideal for global mapping. Likewise, batch to batch variability between RedaC:T cDNA samples casts doubt on relative stoichiometry assessments. Here, we introduce RetraC:T (define) as a method with enhanced ability to detect ac4C in cellular RNA. In brief, RNA is reduced through NaBH4 or the closely related reagent cyanoborohydride (NaCNBH3) followed by cDNA synthesis in the presence of a modified DNA nucleotide, 2-amino-dATP, that preferentially binds to H4-ac4C. Incorporation of the modified dNTP substantially improved C:T mismatch rates and reproducibility, resulting in about 80 percent C:T mismatches at the fully modified site in 18S rRNA. Importantly, amino-dATP did not result in truncated cDNA products nor increase mismatches at other locations, as shown through primer extension assays and nanopore cDNA sequencing, respectively. Thus, modified dNTPs are introduced as a new addition to the toolbox for detecting modified RNA nucleotides at base resolution. Less...