variants with target sequence-selective binding and hydrolyzing activity against 18-bp single-stranded (ss)- nucleic acids. The sequence-selective 3D8 VL variants penetrated into living cells and selectively decreased the amounts of the target mRNAs as well as the proteins expressed by these mRNAs, with minimal eﬀects on oﬀ- target genes. In particular, a Her2/neu-targeting 3D8 VL variant induced apoptotic cell death of Her2- overexpressing cells by down-regulating Her2 expression after cellular internalization. Our results provide a new gene silencing tool mediated by interfering transbody, which would have potential applications in anti-cancer or anti-viral therapies. MATERIALS AND METHODS Materials All oligonucleotides were synthesized from Integrated DNA technologies (Coralville, IA), unless otherwise speciﬁed. Target substrates of 18-bp ss-DNAs and ss- RNAs, G18 (50-GGG GGG GGG GGG GGG GGG-30 for ss-DNA; (G4U)3G3 for ss-RNA) and Her218 (50-AAT TCC AGT GGC CAT CAA-30 for ss-DNA; 50-AAU UCC AGU GGC CAU CAA-30 for ss-RNA), were synthesized with or without 50-biotinylation (12,13). Oﬀ- target 18 bp ss-DNAs with contiguous stretches of single nucleobases, such as T18, C18 and A18, or random sequences N18 (N = A/T/G/C) were also synthesized with or without 50-biotinylation. An oﬀ-target substrate of 18-bp ss-RNA N18 (N = A/U/G/C) was synthesized as above. To construct enhanced green ﬂuorescent protein (EGFP) (the GFP carries two mutations of Phe64Leu and Ser65Thr) reporter plasmid, the target sequence of G18 and Her218 was placed between the ATG start codon and EGFP coding sequence in the pEGFP-N1 plasmid (Clontech), resulting in pG18-EGFP and pHer218-EGFP, respectively. The full-length Her2 cDNA (NCBI accession no. M11730) was subcloned into pcDNA3.1( + ) with NheI and BamHI sites, resulting in pcDNA3-Her2. Her218-siRNA, which has the sequence 50- AAU UCC AGU GGC CAU CAA AdTdT-30, was synthesized at Bioneer Co. (Daejeon, Korea). All other reagents were analytical grade, the details of which are provided in the Supplementary Data. 3D8 VL library construction and screening The gene encoding 3D8 VL derivative 4M, which has four mutations of Q42R, Y49H, W50R and H94A, compared with 3D8 VL wild-type (WT) (NCBI accession no. AAF79129) (12,13), was subcloned in-frame into the yeast surface display plasmid, pCTCON (14). Using 3D8 VL 4M as a template (Supplementary Figure S1), the 3D8 VL library was generated by serial overlapping PCRs to reconstitute full-size products using ﬁve partially overlapping oligonucleotides and two ﬂanking oligo- nucleotides, which were designed to randomize targeted mutation residues at the in C-, C0-, F-strands with the NNB codon. Library construction (Supplementary Figure S1) and library screening against biotinylated- target ss-DNAs (Supplementary Figure S2) were Nucleic Acids Research, 2010, Vol. 38, No. 5 1597 conducted using previously described protocols (14) (also see Supplementary Data for details). Protein preparation and biochemical analyses 3D8 VL WT and HW1 scFv were prepared from the supernatant of bacterial culture, as essentially described previously (12,13,15). After genes encoding 3D8 VL variants, including 4 M, were subcloned from the yeast surface display vector into in frame the bacterial cytosolic expression plasmid of pETdwHis using NheI/BamHI sites (15), they were expressed in Escherichia coli BL21 (DE3). While 4 M expressed solubly in the cytosol of E. coli was puriﬁed using its C-terminus 6 His tag (12,13), isolated 3D8 VL variants expressed dominantly in insoluble form of inclusion body were refolded and puriﬁed as described previously (16). The yield of puriﬁed protein was 2–3 mg out of 1-l ﬂask culture. Protein concentrations were deter- mined using the Bio-Rad protein assay kit. Biochemical analyses of 3D8 VLs, such as DNA- and RNA- hydrolyzing assays on agarose gels (7,13,17) and surface plasmon resonance (SPR) analysis (12,13) were described previously, the details of which are provided in the ﬁgure legends and Supplementary Data. Sequence-speciﬁc nucleic-acid-hydrolyzing assays Sequence-speciﬁc ss-DNA and ss-RNA-hydrolyzing kinetic assays of 3D8 VL WT and its variants were carried out by ﬂuorescence resonance energy transfer (FRET)-based cleavage assay using 18-bp target and oﬀ-target substrates, which were double-labeled with a 6-carboxyﬂuoroscein (FAMTM) at the 50-terminus and a black-hole quencher (BHQ -1) at the 30-terminus (Integrated DNA Technologies) (7,18). For ss-DNA FRET substrates, the following substrates were used: A18, 50-FAM-AAA AAA AAA AAA AAA AAA-BHQ- 1-30; T18, 50-FAM-TTT TTT TTT TTT TTT TTT-BHQ-1- 30; C18, 50-FAM-CCC CCC CCC CCC CCC CCC-BHQ- 1-30; (G4T)3G3, 50-FAM-GGG GTG GGG TGG GGT GGG-BHQ-1-30; Her218, 50-FAM-AAT TCC AGT GGC CAT CAA-BHQ-1-30; N18, 50-FAM-NNN NNN NNN NNN NNN NNN-BHQ-1-30 (N = A/T/G/C). For ss-RNA FRET substrates, the following two substrates were used: Her218, 50-FAM-AAU UCC AGU GGC CAU CAA-BHQ-1-30; N18, 50-FAM-NNN NNN NNN NNN NNN NNN-BHQ-1-30 (N = A/U/G/C). FRET reactions were initiated at 37 C in 96-well Greiner black plate (Sigma-Aldrich) by adding 100 ml of 3D8 VLs (ﬁnal 100 nM) to 100 ml of samples containing various concen- trations of dual-labeled substrates (16 nM–2 mM) in TBS buﬀer with 2 mM MgCl2 for ss-DNAs or without 2 mM MgCl2 for ss-RNAs. Increase of ﬂuorescence intensity due to the DNA/RNA hydrolysis was immediately measured by excitation at 480 and emission at 525 nm for 30 min at 30 s interval in a ﬂuorescence microplate reader (Molecular Devices). Fluorescence did not signiﬁcantly increase over time when 10 mM of the dual-labeled oligonucleotides were incubated in reactions without 3D8 VLs. The raw ﬂuorescence data were corrected for background signal determined in the absence of proteins and then scaled so that all the initial values were the same