A selective and sensitive detection system for 4-thiouridine modification in RNA

4-Thiouridine (s⁴U) is a modified nucleoside, found at positions 8 and 9 in tRNA from eubacteria and archaea. Studies of the biosynthetic pathway and physiological role of s⁴U in tRNA are ongoing in the tRNA modification field. s⁴U has also recently been utilized as a biotechnological tool for analysis of RNAs. Therefore, a selective and sensitive system for the detection of s⁴U is essential for progress in the fields of RNA technologies and tRNA modification. Here, we report the use of biotin-coupled 2-aminoethyl-methanethiosulfonate (MTSEA biotin-XX) for labeling of s⁴U and demonstrate that the system is sensitive and quantitative. This technique can be used without denaturation; however, addition of a denaturation step improves the limit of detection. Thermus thermophilus tRNAs, which abundantly contain 5-methyl-2-thiouridine, were tested to investigate the selectivity of the MTSEA biotin-XX s⁴U detection system. The system did not react with 5-methyl-2-thiouridine in tRNAs from a T. thermophilus tRNA 4-thiouridine synthetase (thiI) gene deletion strain. Thus, the most useful advantage of the MTSEA biotin-XX s⁴U detection system is that MTSEA biotin-XX reacts only with s⁴U and not with other sulfur-containing modified nucleosides such as s²U derivatives in tRNAs. Furthermore, the MTSEA biotin-XX s⁴U detection system can analyze multiple samples in a short time span. The MTSEA biotin-XX s⁴U detection system can also be used for the analysis of s⁴U formation in tRNA. Finally, we demonstrate that the MTSEA biotin-XX system can be used to visualize newly transcribed tRNAs in S. cerevisiae cells.