(Bioneer, Korea) and oligo dT primer (7). The synthesized cDNA was then diluted and used as a template to amplify transcripts speciﬁc for 3D8 VLs, EGFP, Her2, and/or b-actin by PCR with the same number of cycles (30 cycles) (7). The endogenous gene, b-actin, was served to normalize the total amount of mRNA used in each sample (20). The PCR products were then applied to 1% agarose gel electrophoresis followed by ethidium bromide staining. Western blotting Western blotting for cell lysates treated as speciﬁed in ﬁgure legends was performed following the standard procedure, using primary antibodies speciﬁc for target molecules (7,15,19). The appropriate secondary IgG con- jugated to horse radish peroxidase (Zymed Laboratories) was used for developing by chemiluminescence (Amersham Pharmacia Biotech). RESULTS 3D8 VL library design and construction 3D8 VL WT possess intrinsic DNA/RNA-hydrolyzing activity without sequence speciﬁcity in the presence of a divalent metal ion, including Mg2+ and Co2+ (12,13,17). Although the structure of the complex between 3D8 VL WT and DNA/RNA is currently unavailable, the recently resolved 3D8 VL WT structure complexed with a phos- phate mimetics and Co2+ (17) suggests that the residues of Leu33, Tyr49, Trp50, Lys89 and Ser91 are the putative DNA/RNA-hydrolyzing catalytic site. These residues are located in the upper part on one of two b-sheets (Figure 1B), indicating that 3D8 VL might interact with DNA/RNA using the groove formed by the three- stranded b-sheet (C-, C0- and F-strands). The eﬃcient DNA/RNA-hydrolyzing activity of the 3D8 VL WT derivative 4M (Figure 2A and B), which has four substi- tutions of Q42R, Y49H, W50R and H94A in the groove (Supplementary Figure S3) (13,17), supported the rationale of randomizing residues on the C0-, C- and F- strands to isolate DNA/RNA sequence-speciﬁc 3D8 VL variants. We constructed a yeast surface-displayed 3D8 VL library on the template of 4 M by performing successive overlapping PCR mutagenesis, using partially overlapping oligonucleotides designed to introduce random mutations with a degenerate codon NNB at the 15 targeted residues of the C- (35–39 residues), C0- (44–48 residues) and F- strands (84–88 residues) (Figure 1C and Supplementary Figure S1). The NNB codon encodes all 20 amino acids with a reduced stop codon frequency (2.1%) (21). The constructed 3D8 VL library showed the diversity of 8 107, which sparsely sampled the theoretical sequence space (> 3 1019). The initial library with random mutations with 20 possible amino acids at the targeted regions was expressed well on the yeast cell surface (Supplementary Figure S1). Nucleic Acids Research, 2010, Vol. 38, No. 5 1599 Figure 1. (A) Schematic diagrams showing the concept of the interfering transbody. Cell-penetrating antibody (transbody) equipped with sequence-speciﬁc nucleic-acid-hydrolyzing activity penetrates into the cytosol of living cells and preferentially recognizes and hydrolyzes the target mRNA, leading to target gene silencing. (B) Structural char- acteristics of 3D8 VL. Three-dimensional structure of the complex between 3D8 VL WT and Co2+ (gray ball) (PDB code 3BD5) (17). The putative catalytic residues are highlighted and described in detail in the text. Each b-strand is indicated by a diﬀerent color code. (C) 3D8 VL library generation scheme. The library was generated by randomizing 15 putative nucleic-acid binding residues in the groove composed of the C- (35–39 residues), C0- (44–48 residues) and F-strands (84–88 residues) with a degenerate codon of NNB (N = A/ T/G/C, B = C/G/T) based on 3D8 VL 4M as a template (Supplementary Figure S1). Numbering is according to the Kabat def- inition (12). Amino acids and nucleotide bases are indicated in single-letter code according to IUPAC. Isolation of 3D8 VL variants against target 18-bp ss-DNA substrates For model target substrates, we used two 18-bp ss-DNA substrates, contiguous guanine nucleobase (G18) and the Her2/neu-targeting sequence (Her218). The Her218 sequence, corresponding to positions 2391–2408 of the Her2/neu gene, was designed according to an algorithm to search siRNA target sequences (22). The oncogene Her2 is widely overexpressed in many human epithelial tumors, making it an attractive target for anti-cancer agents (22,23). The ss-DNA was employed as a substrate rather than ss-RNA because ss-RNA is highly sensitive to self-hydrolysis. Using two rounds of magnetic activated cell sorting (MACS) followed by four rounds of ﬂuores- cence activated cell sorting (FACS) with a high salt (300 mM NaCl) containing buﬀer and oﬀ-target substrates as competitors to counterselect against non-speciﬁc binders, six clones against G18 and ﬁve against Her218 were isolated and designated 4MG1-6 and 4MH1-5, respectively (Supplementary Figures S2 and S3). The isolated 3D8 VL variants contained 5–15 substitutions out of the 15 targeted residues (Supplementary Figure S3). Biochemical characterization of isolated 3D8 VL variants All of the isolated variants were subcloned in-frame into a bacterial cytoplasmic expression plasmid. While 3D8 VL WT and 4M were expressed solubly in E. coli (12,13), all of the isolated 3D8 VL variants were expressed dominantly in insoluble form of inclusion body. Thus they were refolded and puriﬁed with> 90% purity, as shown in SDS–PAGE analyses (Supplementary Figure