The heptamer sgRNA targeting the human OCT4 mRNA can upregulate the OCT4 expression.

TRUE gene silencing is one of the gene suppression technologies. This technology exploits the enzymatic property of the tRNA 3' processing endoribonuclease tRNase ZL, which is that it can cleave a target RNA under the direction of a small guide RNA (sgRNA). We have been working on the development of therapeutic sgRNAs for hematological malignancies. In the course of an experiment to examine the ability of the heptamer-type sgRNA H15792 targeting the OCT4 mRNA to differentiate human amnion stem cells, we observed unexpectedly that the amnion cells exhibited a morphology resembling initialized cells. Here we investigated the effect of H15792 on human HL60 leukemia cells, and found that H15792 can upregulate the OCT4 expression and the expression of alkaline phosphatase in the cells.

Expanding the utility of heptamer-type sgRNA for TRUE gene silencing.

tRNase Z(L)-utilizing efficacious gene silencing (TRUE gene silencing) is a novel technology for suppressing gene expression. TRUE gene silencing is based on a unique enzymatic property of mammalian tRNase Z(L), which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like or micro-pre-tRNA-like complex formed between the target RNA and artificial small guide RNA (sgRNA). sgRNA is divided into four groups, 5'-half-tRNA, RNA heptamer, hook RNA, and ∼14-nt linear RNA. One of the final destinations of TRUE gene silencing is to generate cancer therapeutic sgRNAs, and from a pharmacological point of view, heptamer-type sgRNA appears to be the most appropriate for this purpose. In this paper, we present two strategies to expand the utility of heptamer-type sgRNA: one is about locked nucleic acid (LNA) modifications of heptamers and the other is about usage of double heptamers. We showed that RNA heptamers with LNA modifications can work as sgRNA in vitro and in vivo. We also demonstrated that two consecutively aligned heptamers can guide target RNA cleavage by human tRNase Z(L) as efficiently as a corresponding 14-nt sgRNA in vitro and that a double heptamer can work much better than a 14-nt sgRNA in vivo.