title: Optimization of shRNA inhibitors by variation of the terminal loop sequence.
abstract: Gene silencing by RNA interference (RNAi) can be achieved by intracellular expression of a short hairpin RNA (shRNA) that is processed into the effective small interfering RNA (siRNA) inhibitor by the RNAi machinery. Previous studies indicate that shRNA molecules do not always reflect the activity of corresponding synthetic siRNAs that attack the same target sequence. One obvious difference between these two effector molecules is the hairpin loop of the shRNA. Most studies use the original shRNA design of the pSuper system, but no extensive study regarding optimization of the shRNA loop sequence has been performed. We tested the impact of different hairpin loop sequences, varying in size and structure, on the activity of a set of shRNAs targeting HIV-1. We were able to transform weak inhibitors into intermediate or even strong shRNA inhibitors by replacing the loop sequence. We demonstrate that the efficacy of these optimized shRNA inhibitors is improved significantly in different cell types due to increased siRNA production. These results indicate that the loop sequence is an essential part of the shRNA design. The optimized shRNA loop sequence is generally applicable for RNAi knockdown studies, and will allow us to develop a more potent gene therapy against HIV-1.
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, Amsterdam, The Netherlands.
authors.forename: Nick C T
authors.initials: NC
authors.lastname: Schopman
authors.forename: Ying Poi
authors.initials: YP
authors.lastname: Liu
authors.forename: Pavlina
authors.initials: P
authors.lastname: Konstantinova
authors.forename: Olivier
authors.initials: O
authors.lastname: ter Brake
authors.forename: Ben
authors.initials: B
authors.lastname: Berkhout
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001688:Biological Products / Q000235:genetics / Q000494:pharmacology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D015497:HIV-1 / Q000187:drug effects; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D001688:Biological Products; D034741:RNA, Small Interfering