Elimination of specific miRNAs by naked 14-nt sgRNAs.

Abstract

tRNase Z(L)-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase Z(L), which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5'-half-tRNA, RNA heptamer, hook RNA, and ~14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase Z(L) cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ~50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing.