title: Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.
abstract: Short interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene knockdown in mammalian cells. To clarify the position-dependent functions of ribonucleotides in siRNA, siRNAs with various DNA substitutions were constructed. The following could be simultaneously replaced with DNA without substantial loss of gene-silencing activity: the seed arm, which occupies positions 2-8 from the 5'end of the guide strand; its complementary sequence; the 5'end of the guide strand and the 3'overhang of the passenger strand. However, most part of the 3' two-thirds of the guide strand could not be replaced with DNA, possibly due to binding of RNA-recognition proteins such as TRBP2 and Ago2. The passenger strand with DNA in the 3'end proximal region was incapable of inducing off-target effect. Owing to lesser stability of DNA-RNA hybrid than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases, incapable to exert unintended gene silencing due to seed sequence homology. Thus, it may be possible to design DNA-RNA chimeras which effectively silence mammalian target genes without silencing unintended genes.
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan. ktei@biochem.s.u-tokyo.ac.jp
authors.forename: Kumiko
authors.initials: K
authors.lastname: Ui-Tei
authors.forename: Yuki
authors.initials: Y
authors.lastname: Naito
authors.forename: Shuhei
authors.initials: S
authors.lastname: Zenno
authors.forename: Kenji
authors.initials: K
authors.lastname: Nishi
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamato
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Takahashi
authors.forename: Aya
authors.initials: A
authors.lastname: Juni
authors.forename: Kaoru
authors.initials: K
authors.lastname: Saigo
mesh_terms: D020856:AT Rich Sequence; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000737:chemistry*; D003854:Deoxyribonucleotides / Q000737:chemistry; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D023281:Genomics; D006801:Humans; D050814:Octamer Transcription Factor-3 / Q000037:antagonists & inhibitors; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012265:Ribonucleotides / Q000737:chemistry; D014162:Transfection
chemical_list: D003854:Deoxyribonucleotides; D050814:Octamer Transcription Factor-3; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D012265:Ribonucleotides; D004247:DNA