title: Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi.
abstract: Artificial microRNA (amiRNA) sequences embedded in natural microRNA (miRNA) backbones have proven to be useful tools for RNA interference (RNAi). amiRNAs have reduced off-target and toxic effects compared to other RNAi-based methods such as short-hairpin RNAs (shRNA). amiRNAs are often less effective for knockdown, however, compared to their shRNA counterparts. We screened a large empirically-designed amiRNA set in the synthetic inhibitory BIC/miR-155 RNA (SIBR) scaffold and show common structural and sequence-specific features associated with effective amiRNAs. We then introduced exogenous motifs into the basal stem region which increase amiRNA biogenesis and knockdown potency. We call this modified backbone the enhanced SIBR (eSIBR) scaffold. Using chained amiRNAs for multi-gene knockdown, we show that concatenation of miRNAs targeting different genes is itself sufficient for increased knockdown efficacy. Further, we show that eSIBR outperforms wild-type SIBR (wtSIBR) when amiRNAs are chained. Finally, we use a lentiviral expression system in cultured neurons, where we again find that eSIBR amiRNAs are more potent for multi-target knockdown of endogenous genes. eSIBR will be a valuable tool for RNAi approaches, especially for studies where knockdown of multiple targets is desired.
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA.
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Fowler
authors.affiliation: Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA.
authors.forename: Carly
authors.initials: C
authors.lastname: Williams
authors.affiliation: Institute for Bioinformatics and Evolutionary Studies, University of Idaho, Moscow, ID 83844, USA.
authors.forename: Alida T
authors.initials: AT
authors.lastname: Gerritsen
authors.affiliation: Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA pwash@uoneuro.uoregon.edu.
authors.forename: Philip
authors.initials: P
authors.lastname: Washbourne
mesh_terms: D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000235:genetics*; D061251:Primary Cell Culture; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: C524942:MIRN155 microRNA, human; D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins