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

(A) Sequences of Linear-s⁴U and Linear-U RNAs. Linear-s⁴U contains s⁴U only at position 9 from 5′-end. Linear-U contains U instead of s⁴U. (B) When MTSEA biotin-XX is reacted with s⁴U, the absorbance at 330 nm derived from s⁴U disappears. To determine the reaction time, decrease of absorbance at 330 nm in a reaction mixture containing Linear-s⁴U and MTSEA biotin-XX was monitored. (C) Comparison of the reactivity of Linear-s⁴U and Linear-U RNAs toward MTSEA biotin-XX. Both RNAs were treated with MTSEA biotin-XX and then fixed onto a nylon membrane filter. RNAs were visualized by methylene blue staining (upper). The bound MTSEA biotin-XX was detected by chemiluminescence (lower). (D) Relationship between the amounts of RNAs and the chemiluminescence intensity of spots showed linearity from 0 pmol to 100 pmol RNAs. Chemiluminescence derived from the Linear-U samples was undetectable. Relative intensity is defined so that the chemiluminescence derived from the 50 pmol Linear-s⁴U sample is expressed as 1.00.