Abstract
Dicer-substrate siRNA (DsiRNA) was a useful tool for sequence-specific gene silencing. DsiRNA was proposed to have increased efficacy via RNAi gene silencing, but the molecular mechanism underlying the increased efficacy is not precise. We designed the tetra-looped DsiRNA as the tetra-looped RNAs have been reported more stable structure and increased binding efficiency with RNA and protein. To gain a deeper understanding of the Dicer function of DsiRNA, we knocked out Dicer in the HCT116 cell line and analyzed the efficacy of various Dicer substrates on RNAi gene silencing activity. Tetra-looped DsiRNA demonstrated increased efficacy of gene silencing Dicer expressing cells with activity favoring the guide strand. The gene silencing activity of all DsiRNAs was reduced in Dicer knockout cells. Thus, this study allows us to understand the Dicer function of key RNAi silencing and provides valuable resources for RNAi research and applications.
The abbreviations used are
- DsiRNA
- Dicer-substrate siRNA
- rRNA
- ribosomal RNA
- dsRNAs
- double-stranded RNAs
- siRNAs
- small interfering 21-22bp RNAs
- RISC
- RNA-induced silencing complex
- miRNA
- microRNA
- pri-miRNA
- hairpin-containing primary transcripts
- shRNAs
- short hairpin RNAs
- DExD
- DEAD-like helicases domain
- TRBP
- transactivation response element RNA-binding protein
- WT
- wild-type
- RNAi
- RNA interference
- hnRNPH1
- heterogeneous nuclear ribonucleoprotein H
- TL
- tetra-loop
- GFP
- green fluorescent protein
- gDNA
- genomic DNA
- sgRNA
- single-guide RNA