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. 2015;12(4):447-56.
doi: 10.1080/15476286.2015.1022024.

Toward optimization of AgoshRNA molecules that use a non-canonical RNAi pathway: variations in the top and bottom base pairs

Elena Herrera-Carrillo  1 , Alex Harwig, Ben Berkhout
Affiliations

Toward optimization of AgoshRNA molecules that use a non-canonical RNAi pathway: variations in the top and bottom base pairs

Elena Herrera-Carrillo et al. RNA Biol. 2015.

Abstract

Short hairpin RNAs (shRNAs) are widely used for gene knockdown by inducing the RNA interference (RNAi) mechanism. The shRNA precursor is processed by Dicer into small interfering RNAs (siRNAs) and subsequently programs the RNAi-induced silencing complex (RISC) to find a complementary target mRNA (mRNA) for post-transcriptional gene silencing. Recent evidence indicates that shRNAs with a relatively short basepaired stem bypass Dicer to be processed directly by the Ago2 nuclease of the RISC complex. We named this design AgoshRNA as these molecules depend on Ago2 both for processing and subsequent silencing activity. This alternative AgoshRNA processing route yields only a single active RNA strand, an important feature to restrict off-target effects induced by the passenger strand of regular shRNAs. It is therefore important to understand this novel AgoshRNA processing route in mechanistic detail such that one can design the most effective and selective RNA reagents. We performed a systematic analysis of the optimal base pair (bp) composition at the top and bottom of AgoshRNA molecules. In this study, we document the importance of the 5' end nucleotide (nt) and a bottom mismatch. The optimized AgoshRNA design exhibits improved RNAi activity across cell types. These results have important implications for the future design of more specific RNAi therapeutics.

Keywords: RNA processing; agoshRNA; argonaute2; dicer; shRNA.

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Figures

Figure 1.
Figure 1.
Characteristics of the traditional shRNA and novel AgoshRNA design. (A) Secondary structure of a shRNA and AgoshRNA molecule. The shRNA is processed by Dicer (▸◂) into an siRNA duplex of ≥20 bp with 3′ overhangs that is loaded into RISC. The passenger strand (white arrow) is cleaved and subsequently degraded, the guide strand (black arrow) is active in RNAi-silencing. The shorter AgoshRNA duplex (stem 17–19 bp) is not recognized by Dicer and consequently processed by Ago2. The AgoshRNA duplex is cleaved (◂) on the 3′ side between bp 10 and 11 to yield a single RNA molecule of ∼33 nt (gray arrow) that instructs Ago2 for RNAi-silencing. (B) Luc reporter constructs with sense and antisense target sequences. The Luc-sense reporter scores canonical shRNA guide activity, the Luc-antisense reporter scores both shRNA passenger and AgoshRNA activity.
Figure 2.
Figure 2.
Design of AgoshRNAs with variation in the 2 top base pairs. AgoshRT5 and AgoshPol47 with 19 bp stem and 5 nt loop (19/5) were used as wild-type (WT) backbones. The encoded guide sequence is boxed. The top bp was modified (mutated bp in black).
Figure 3.
Figure 3.
Knockdown activity and processing of AgoshRT5 variants. (A) The knockdown activity of the different AgoshRNA variants was determined by co-transfection with a luciferase reporter containing either the sense- or antisense-target sequence. HEK293T cells were co-transfected with 100 ng of the respective firefly luciferase reporter plasmid, 1 ng renilla luciferase plasmid as internal control and 1, 5 or 25 ng of the corresponding shRNA construct. An unrelated shRNA (shNef) served as negative control, this activity was set at 100% luciferase expression. We performed 3 independent transfections, each in duplicate, and standard deviations were calculated. (B) Processing of the 3′ strand (upper panel) and 5′ strand (lower panel) of the AgoshRT5 variants was analyzed by RNA gel blot. The AgoshRNAs varied in the 2 top bp. HEK293T cells were transfected with 5 μg of the indicated constructs. Size markers were included in the far right lane (length indicated in nt). An unrelated shRNA (shNef) was included as negative control. The regular shRNA ∼21 nt products are marked and * indicates the AgoshRNA ∼33 nt products.
Figure 4.
Figure 4.
Knockdown activity and processing of AgoshPol47 variants. (A) The knockdown activity of the guide strand on Luc-sense (upper panel) and passenger strand on Luc-antisense (lower panel) of the AgoshPol47 was determined by co-transfection of a luciferase reporter encoding the sense and antisense target sequence, respectively, in HEK293T cells. We performed 3 independent transfections, each in duplicate, and standard deviations were calculated. (B) Total RNA was analyzed by northern blot for processing products derived from the 3′ strand (upper panel) and 5′ strand (lower panel). Size markers are indicated on the right. The regular shRNA ∼21 nt products are marked and * indicates the AgoshRNA ∼30 nt products. See Figure 3 for more details.
Figure 5.
Figure 5.
Design of shRNA and AgoshRNA mutants with variation in the bottom bp. shRNAs (21/5) and AgoshRNAs (19/5) based on the RT5 and Pol47 inhibitors were used as WT backbones. The bottom bp was substituted by the mismatches A·C and U·C (mutated nt in black).
Figure 6.
Figure 6.
Knockdown activity of the A·(C) and U·(C) AgoshRNA variants. The knockdown activity of the guide strand on Luc-sense (upper panel) and the passenger strand on Luc-antisense (lower panel) was determined by co-transfection of a luciferase reporter encoding the sense (top) and antisense (bottom) target sequence in HEK293T cells. We performed 3 independent transfections, each in duplicate, and standard deviations were calculated. See Figure 3 for additional details.
Figure 7.
Figure 7.
Processing of shRNAs and AgoshRNAs is influenced by the bottom base pair. (A, B) RT5 and (C, D) Pol47 variants were analyzed by RNA gel blot. HEK293T cells were transfected with 5 μg of the corresponding RNA constructs. The shRNAs and AgoshRNAs (21/5 and 19/5) varied in bottom bp (WT is A-U for RT5 and G-C for Pol47, and mutants A·C or U·C). Total RNA was isolated and analyzed by northern blot using an LNA probe to detect processing products derived from the 3′ side (A, C) and the 5′ side (B, D). Size markers were included (length indicated in nt). An unrelated shRNA (shNef) was included as negative control. The regular shRNA ∼21 nt products are marked and * indicates the AgoshRNA ∼30 nt products.
Figure 8.
Figure 8.
Improved AgoshRNA activity with a bottom A·(C) mismatch. C33A and Vero cells were co-transfected with 100 ng of the respective firefly luciferase reporter plasmid, 1 ng renilla luciferase plasmid as internal control and 1, 5 or 25 ng of the AgoshPol47 variants (WT with G-C and mutants A·C or U·C). An unrelated shRNA (shNef) served as negative control for which the activity obtained was set at 100% luciferase expression. We performed 3 independent transfections, each in duplicate, and standard deviations were calculated.
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