Cellular siRNA delivery mediated by a cell-permeant RNA-binding protein and photoinduced RNA interference.
HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the U1A RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5'-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. We refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.
Endoh
Tamaki
T
Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Sisido
Masahiko
M
Ohtsuki
Takashi
T
D016915:AIDS Vaccines / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D016503:Drug Delivery Systems; D024202:Electrophoretic Mobility Shift Assay; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D066246:ErbB Receptors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D010777:Photochemistry; D034622:RNA Interference / Q000187:drug effects / Q000528:radiation effects; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D016915:AIDS Vaccines; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C421254:human immunodeficiency virus type 1 Tat vaccine; D049452:Green Fluorescent Proteins; D066246:ErbB Receptors