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.