alexa The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA.
Biochemistry

Biochemistry

Biochemistry & Analytical Biochemistry

Author(s): Saayman S, Barichievy S, Capovilla A, Morris KV, Arbuthnot P,

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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.
This article was published in PLoS One and referenced in Biochemistry & Analytical Biochemistry

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