1596–1609 Nucleic Acids Research, 2010, Vol. 38, No. 5 doi:10.1093/nar/gkp1145 Published online 9 December 2009 Gene silencing by cell-penetrating, sequence- selective and nucleic-acid hydrolyzing antibodies Woo-Ram Lee1, Ji-Young Jang2, Jeong-Sun Kim3, Myung-Hee Kwon2 and Yong-Sung Kim1,* 1Department of Molecular Science and Technology, Ajou University, Suwon 443-749, 2Department of Microbiology, Ajou University School of Medicine, Suwon 442-721 and 3Department of Chemistry, Chonnam National University, Gwangju, 500-757, Korea Received September 10, 2009; Revised November 17, 2009; Accepted November 18, 2009 ABSTRACT Targeting particular mRNAs for degradation is a fascinating approach to achieve gene silencing. Here we describe a new gene silencing tool exploit- ing a cell-penetrating, nucleic-acid hydrolyzing, single-domain antibody of the light-chain variable domain, 3D8 VL. We generated a synthetic library of 3D8 VL on the yeast surface by randomizing residues located in one of two b-sheets. Using 18-bp single-stranded nucleic acids as target substrates, including the human Her2/neu-targeting sequence, we selected 3D8 VL variants that had 100–1000-fold higher affinity and 2–5-fold greater selective hydrolyzing activity for target substrates than for off targets. 3D8 VL variants effi- ciently penetrated into living cells to be accumula- ted in the cytosol and selectively decreased the amount of target sequence-carrying mRNAs as well as the proteins encoded by these mRNAs with minimal effects on off-target genes. In particular, one 3D8 VL variant targeting the Her2 sequence showed more efficient downregulation of Her2 expression than a small-interfering RNA targeting the same Her2 sequence, resulting in apoptotic cell death of Her2-overexpressing breast cancer cells. Our results demonstrate that cell-penetrating 3D8 VL variants with sequence-selective, nucleic- acid-hydrolyzing activity can selectively degrade target mRNAs in the cytosol, providing a new gene silencing tool mediated by antibody. INTRODUCTION Gene silencing by targeting speciﬁc genes for degradation, particularly at the mRNA level, is an invaluable tool for gene function analysis and a powerful therapeutic strategy for human diseases, including cancer and viral infections (1,2). Nucleic-acid based approaches that speciﬁcally rec- ognize and hydrolyze particular regions of targeted RNA have been developed for this purpose, including antisense oligonucleotides and interference RNAs (RNAi) (1,2). The RNAi technique is now readily available, in which 21–23 bp double-stranded (ds)-RNAs, so-called small interfering RNAs (siRNA), cause sequence-speciﬁc degra- dation of complementary mRNAs (3,4). Although siRNAs can be directly designed for the target sequence based on Watson–Crick base pairing, their practical appli- cation has been limited by several factors, including cellular delivery, nuclease susceptibility and oﬀ-target eﬀects (1–4). Another approach for degrading cytosolic RNAs is the use of protein-based RNases (5) and DNA/RNA- hydrolyzing monoclonal antibodies (mAbs) (6,7), which can penetrate into living cells and degrade cytosolic RNAs. However, these approaches lack high sequence- speciﬁcity, leading to signiﬁcant cytotoxicity (5–7). Although some RNases have been fused with peptides that confer both cell-penetrating and sequence-speciﬁc recognition abilities (8,9), these fused RNases cannot be used as a general gene-silencing tool for other genes. As an alternative approach to conventional techniques, we here describe proof-of-concept for an ‘interfering transbody’ technology, in which a cell-penetrating antibody (transbody) (10,11) equipped with sequence- speciﬁc, nucleic-acid-hydrolyzing activity selectively rec- ognizes and hydrolyzes the target mRNA in the cytosol of living cells, leading to gene silencing (Figure 1A). Recently we reported a sequence-non-speciﬁc DNA/ RNA-hydrolyzing single-domain antibody of the light- chain variable domain, 3D8 VL (7,12,13), which has cell-penetrating ability. Here, from a yeast surface- displayed 3D8 VL library generated by randomizing potential base-interacting residues, we isolated 3D8 VL *To whom correspondence should be addressed. Tel: +82 31 219 2662; Fax: +82 31 219 2394; Email: kimys@ajou.ac.kr The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors. The Author(s) 2009. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.