RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future. 1. Introduction RNA interference (RNAi) is an evolutionarily conserved, gene-silencing mechanism that is triggered by double-stranded RNA (dsRNA). Two types of small RNA—namely, small interfering RNA (siRNA) and microRNA (miRNA)—are central players in RNAi. Both siRNAs and miRNAs regulate gene expression by annealing to mRNA sequence elements that are fully or partially complementary [1, 2]. Since transfected synthetic siRNAs were shown to induce RNAi in mammalian cells , they have been widely used to decipher gene function through suppression of gene expression, and they have also attracted attention because of their potential for therapeutic applications [4, 5]. miRNAs are a phylogenetically conserved family of endogenous small RNAs that play important roles in a wide variety of biological functions, including cell differentiation, tumor genesis, apoptosis, and metabolism [1, 2, 6, 7]. miRNAs are initially generated as long primary transcripts (pri-miRNA) that are processed in the nucleus by the enzyme complexes Drosha and DiGeorge Critical Region 8 (DGCR8) to a 70–90？nt stem-loop structure called pre-miRNA. The pre-miRNA is then exported to the cytoplasm. There, the exported pre-miRNA or exogenous dsRNA is cleaved by the enzyme Dicer into a ~22-nucleotide (nt) duplex known as miRNA or siRNA, respectively. The duplex is then incorporated into the RNA-induced silencing complex (RISC). After removing one strand called the passenger strand, the remaining strand,
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