title: The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA.
abstract: RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs.
authors.affiliation: Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of Witwatersrand, Johannesburg, South Africa.
authors.forename: Sheena
authors.initials: S
authors.lastname: Saayman
authors.forename: Samantha
authors.initials: S
authors.lastname: Barichievy
authors.forename: Alexio
authors.initials: A
authors.lastname: Capovilla
authors.forename: Kevin V
authors.initials: KV
authors.lastname: Morris
authors.forename: Patrick
authors.initials: P
authors.lastname: Arbuthnot
authors.forename: Marc S
authors.initials: MS
authors.lastname: Weinberg
mesh_terms: D019380:Anti-HIV Agents / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D004258:DNA Polymerase III / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000096:biosynthesis / Q000235:genetics*; D012367:RNA, Viral / Q000378:metabolism; D014162:Transfection; D014779:Virus Replication
chemical_list: D019380:Anti-HIV Agents; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D012367:RNA, Viral; C405486:U6 small nuclear RNA; D004258:DNA Polymerase III