Ganoderma lucidum (Ganoderma) is a famous Chinese herbal medicine which has been used clinically for thousands of years in China. Despite numerous studies on
triterpenes and
polysaccharides, the bioactivity of RNAs abundant in Ganoderma remains unknown. Here, based on LC-MS techniques,
dihydrouracil,
5-methyluridine (m5U) and
pseudouridine were identified at position 19, 52 and 53 of a new
tRNAIle(GAU) which was isolated as the most abundant
tRNA species in Ganoderma, and is the first purified
tRNA from fungus. Cytotoxic screening of
tRNA-half (t-half) and
tRNA fragment (tRF) derived from this
tRNA, as well as their mimics (t-half or tRF as antisense strand), demonstrated that the double-stranded form, i.e., tRF and t-halve mimics, exhibited stronger cytotoxicity than their single-stranded form, and the cytotoxicity of t-half mimic is significantly stronger than that of tRF mimic. Notably, the cytotoxicity of 3'-t-half mimic is not only much more potent than that of
taxol, but also is much more potent than that of ganoderic
acids, the major bioactive components in Ganoderma. Furthermore, 3'-t-half mimic_M2 (m5U modified) exhibited significantly stronger cytotoxicity than unmodified 3'-t-half mimic, which is consistent with the computational simulation showing that m5U modification enhances the stability of the tertiary structure of 3'-t-half mimic. Overall, the present study not only indicates t-halves are bioactive components in Ganoderma which should not be neglected, but also reveals an important role of post-transcriptional modification on
tRNA in its fragments' cytotoxicity against
cancer cells, which benefits the design and development of RNAi drugs from natural resource.