expressed antigens (15,19,28). The targeting of intracellular proteins has been achieved by intrabodies (11,28,29). However, intrabodies have never been exploited to knockdown speciﬁc proteins at the post- transcriptional mRNA level. Here, we have developed target sequence-selective, hydrolyzing, single-domain antibodies of 3D8 VL variants that can penetrate into living cells and accumulate mostly in the cytosol. These cell-penetrating antibodies (transbodies) recognize and degrade targeted mRNAs, but not proteins, resulting in targeted gene silencing. We engineered in vitro the sequence non-speciﬁc DNA/RNA-hydrolyzing 3D8 VL, which has the typical immunoglobulin fold of b-sandwich, into target sequence-selective hydrolyzing variants by randomizing one-sided b-sheet residues. In vitro engineering of anti- DNA/RNA antibodies that speciﬁcally recognize and hydrolyze speciﬁc DNA/RNA antigens has never been reported, although an RNA-binding Fab has been engi- neered to speciﬁcally recognize the targeted RNA tertiary structure (30) and sequence-speciﬁc DNA binding antibodies have been raised in vivo from mice (31,32). Our results suggest that proteins with b-sheet structure can be used as a scaﬀold to engineer sequence-speciﬁc ss-DNA/RNA binding proteins, as a-helical zinc-ﬁnger protein does in speciﬁc-speciﬁc ds-DNA-binding proteins (33). However, random mutations introduced into the highly conserved framework regions of 3D8 VL, which are otherwise buried in VH–VL interface in IgG format, might induce immunogenic response in vivo. Even though the 18-bp ss-DNAs were employed as screening substrates rather than ss-RNAs due to the higher nucleolytic stability, the selected 3D8 VL variants showed very similar sequence-selective binding and hydrolyzing activities for the targeted sequences in ss- RNA form to those in ss-DNA form in vitro (Table 1 and 2), resulting in the preferential hydrolysis of mRNAs carrying the target sequences in the cytosol of cells. In DNA/RNA–protein interactions, stacking and electrostatic interactions play a key role in providing aﬃnity, whereas hydrogen bonds between nucleobase and amino acids dominantly contribute to the sequence- speciﬁcity as well as aﬃnity (34). The rank orders of nucleobase–amino acid type correlations have shown strong similarities between the DNA and RNA cases (34,35), suggesting the minor diﬀerences between ss- DNA and ss-RNA, including thymine (5-methyluracil) and deoxyribose in DNA in place of uracil and ribose in RNA, do not signiﬁcantly aﬀect the sequence speciﬁcity. Thus, the similar preferential recognition of 3D8 VL variants to their target sequences in either ss-DNA or ss- RNA form suggests that hydrogen bonds between nucleobase and amino acids mainly contribute to their sequence-speciﬁcity. No common patterns of amino acid substitutions were observed among 3D8 VL variants isolated against the same substrates, G18 and Her218 (Supplementary Figure S3), suggesting that substituted residues at each position may be involved in more subtle, context-dependent inter- actions with the target sequences, like the zinc ﬁnger-DNA recognition (33). While random mutations without bias to Nucleic Acids Research, 2010, Vol. 38, No. 5 1607 particular amino acids at the targeted residues were observed in the initial library, most of substitutions in the selected 3D8 VL variants occurred with hydrophilic residues (Arg, Asn, Gln, Glu, Ser, Lys, Asp and His), which could readily contribute to base-speciﬁc recognition by hydrogen bonds (34). Particularly, the selected 3D8 VL variants showed strong preference for Arg substitution at many residues facing outward, particularly for G18-selec- tive 3D8 VL variants (Supplementary Figure S3). Arg is the most common amino acid interacting with guanine base, the pair of which can form up to four hydrogen bonds, in the analysis of amino acid-base contacts in protein–RNA complexes (34,35). Detailed analysis of the contribution of each substitution to the preferential inter- action with target sequences should be waited until the complex structures of 3D8 VL variants with target substrates are obtained. Many anti-DNA/RNA hydrolyzing antibodies, pre- ferentially found in humans and mice with autoimmune diseases, have been reported to have the ability to pene- trate into living cells (6,36–39). Though detailed structural and mechanistic bases how they can cross the plasma membrane of cells remain to be resolved, many studies have suggested that their common feature of possessing a large number of positively-charged amino acids, such as Arg and Lys, in the complementarity-determining regions (CDRs) of VH and/or VL domains due to their antigen binding properties, can be attributed to their internalizing capacity (6,36–39). Thus, like cell-penetrating peptides, non-speciﬁc electrostatic interactions of anti-DNA/ RNA antibodies with negatively charged cell surface matrix, such as heparan sulfate proteoglycans, have been proposed to contribute to their cell-penetrating activity without cell-type speciﬁcity (40,41). 3D8 VL WT and its variants eﬃciently internalized into the cytosol of living cells most likely by the caveolae/lipid raft-mediated endocytic pathway (Figure 4), like 3D8 scFv (7). Three- dimensional structural analysis of 3D8 VL (17) reveals a continuous patch clustered with positively-charged amino acids composed of Arg27f, Arg29 and Lys30 in CDR1, which is also conserved in the selected 3D8 VL variants (Supplementary Figure S3). This unusual cationic property of 3D8 VLs might explain their cellular internal- ization capacity, like other cell-penetrating anti-DNA/ RNA antibodies (37–41). Cytoplasmic accumulation of cellular internalized 3D8 VL variants makes it possible to induce gene silencing of targeted genes, like RNAi-mediated gene silencing that primarily occurs in the cytosol (1–3). The knockdown eﬃciencies of the 3D8 VL variants ranged from 10–100% and increased with the post-treatment period. These ranges are similar to those achieved by conventional siRNA techniques (1–3). Her218-selective 4MH2 induced apoptotic cell death for Her2-overexpressing tumor cells by down-regulating Her2 expression (Figure 6 and 7), indicating that it may have potential as an anti-cancer agent. Compared with Her218-siRNA, the extent to which 4MH2 could knock-down Her2 over a short period ( 2 h) of post-treatment (Figure 6), both at the mRNA and protein level, was remarkable. 3D8 VLs in HeLa and SK-BR-3 cells were diﬀused throughout the