Cellular siRNA delivery using TatU1A and photo-induced RNA interference.
RNA interference (RNAi)-mediated silencing of specific genes represents a powerful tool for analyzing protein function. It also has profound biotechnological applications for cellular engineering and therapeutics. However, it is necessary to have a method that controls RNAi in response to artificially regulated stimulation. We designed a fluorescently labeled carrier protein to deliver short hairpin RNA (shRNA) with activity that could be regulated via photostimulation. We constructed a cell-permeable RNA-binding protein (RBP) by fusing the U1A RBP and a HIV-1 Tat peptide, which was labeled with an Alexa Fluor 546 fluorophore (TatU1A-Alexa). TatU1A-Alexa bound specifically to shRNA, which contains a U1A-binding sequence. The TatU1A-Alexa/shRNA complex was then internalized into cells via an endocytotic pathway and redistributed from endosomes to the cytosol by photostimulation, which induced RNAi-mediated gene silencing. This successive strategy was termed CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi).
Endoh
Tamaki
T
Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Ohtsuki
Takashi
T
D000818:Animals; D002460:Cell Line; D004705:Endocytosis; D018462:Photobiology / Q000379:methods*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000302:isolation & purification
D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus