Gene silencing by cell-penetrating, sequence-selective and nucleic-acid hydrolyzing antibodies.
Targeting particular mRNAs for degradation is a fascinating approach to achieve gene silencing. Here we describe a new gene silencing tool exploiting 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 beta-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 approximately 100-1000-fold higher affinity and approximately 2-5-fold greater selective hydrolyzing activity for target substrates than for off targets. 3D8 VL variants efficiently penetrated into living cells to be accumulated 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.
Lee
Woo-Ram
WR
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Korea.
Jang
Ji-Young
JY
Kim
Jeong-Sun
JS
Kwon
Myung-Hee
MH
Kim
Yong-Sung
YS
D017151:Antibodies, Catalytic / Q000737:chemistry / Q000378:metabolism*; D017209:Apoptosis; D001483:Base Sequence; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D055785:Gene Knockdown Techniques; D015723:Gene Library; D006801:Humans; D006868:Hydrolysis; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D017151:Antibodies, Catalytic; D004277:DNA, Single-Stranded; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human