1608 Nucleic Acids Research, 2010, Vol. 38, No. 5 cytosol within 2 h of medium treatment, with little accu- mulation in the nucleus, similar to 3D8 scFv (7). The eﬃ- cient gene silencing eﬀects achieved immediately after cellular internalization of 3D8 VLs distinguishes 3D8 VLs from siRNAs which require at least 12–18 h for eﬀec- tive gene silencing (1–3). 3D8 VL variants exerted signiﬁcant cytotoxicity ( 30– 40% after 72 h) compared with siRNA (< 10% after 72 h). The selected 3D8 VL variants showed moderate binding aﬃnities of KD 10 7–10 8 M with the target substrates and only 2–5-fold greater selective hydrolyzing activities for the target sequences than oﬀ-targets (Table 1 and 2), which is rather low target speciﬁcity compared with other RNA–protein speciﬁc interactions (30,34,35). Structural features and molecular docking analysis revealed that 3D8 VLs may interact with only 5–7 bases of ss-RNA. This suggests that the limited target sequence recognition caused non-speciﬁc degradation of other cytosolic RNAs (7), resulting in more cytotoxicity than the 19-bp-targeting siRNA. To minimize oﬀ-target eﬀects, modular units of 3D8 VL variants could be linked to confer higher speciﬁcity and aﬃnity for long RNA sequences of up to 12–18 bp, as has previously been done for zinc-ﬁnger proteins (33). Interfering transbody-mediated gene silencing may oﬀer several advantages over conventional nucleic-acid based approaches (1–4,10,11), including cell-penetrating ability without the need for exogenous carriers, RNA deg- radation without external RNases, no nuclease suscepti- bility, and immediate gene-silencing eﬀects after cellular internalization. These distinct features make interfering transbody an attractive alternative to nucleic-based approaches for modulating gene expression in functional genomics or in therapeutic approaches to diseases such as cancer and viral infections. 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