Figure 3:

Significance of inefficient chemistry toward length bias in s⁴U metabolic labeling experiments. (A) Chemistry of activated disulfide reactivity with s⁴U. When HPDP-biotin is used as the activated disulfide, any biotin-s⁴U (bio-s⁴U) product results in a more activated disulfide due to the electron-poor pyrimidine ring of s⁴U, therefore favoring the reverse rather than the forward biotinylation reaction. In contrast, MTS-biotin reacts irreversibly with s⁴U to form the bio-s⁴U product under the reaction conditions, and the covalent disulfide bond can only be reversed under reducing conditions. (B) Schematic of s⁴U-RNA enrichment with HPDP- and MTS-biotin. Efficient activated disulfide reactivity of MTS-biotin results in greater yields of s⁴U-RNA and alleviates potential biases toward longer RNAs with more uridines.