title: Design and Experimental Validation of Small Activating RNAs Targeting an Exogenous Promoter in Human Cells.
abstract: It is increasingly practical to co-opt many native cellular components into use as elements of synthetic biological systems. We present the design and experimental investigation of the first exogenous genetic construct to be successfully targeted by RNA activation, a phenomenon whereby small double-stranded RNAs increase gene expression from sequence-similar promoters by a mechanism thought to be related to that of RNA interference. Our selection of activating RNA candidates was informed by a custom-written computer program designed to choose target sites in the promoter of interest according to a set of empirical optimality criteria drawn from prior research. Activating RNA candidates were assessed for activity against two exogenously derived target promoters, with successful candidates being subjected to further rounds of validation as a precaution against potential off-target effects. A genetic platform was assembled that allowed activating RNA candidates to be simultaneously screened both for positive activity on the target reporter gene and for possible nonspecific effects on cell metabolism. Several candidate sequences were tested to appraise the utility of this platform, with the most successful achieving a moderate activation level with minimal off-target effects.
authors.affiliation: Department of Physics, University of Toronto , 60 St. George Street, Toronto, Ontario M5S 1A7, Canada.|Department of Chemical and Physical Sciences, University of Toronto Mississauga , 3359 Mississauga Road, Mississauga, Ontario L5L 1C6, Canada.|Impact Centre, University of Toronto , 112 College Street, Toronto, Ontario M5G 1A7, Canada.
authors.forename: Edouard A
authors.initials: EA
authors.lastname: Harris
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto , 160 College Street, Toronto, Ontario M5E 3E1, Canada.
authors.forename: Alla
authors.initials: A
authors.lastname: Buzina
authors.affiliation: Department of Molecular Genetics, University of Toronto , 160 College Street, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
authors.affiliation: Department of Physics, University of Toronto , 60 St. George Street, Toronto, Ontario M5S 1A7, Canada.|Department of Chemical and Physical Sciences, University of Toronto Mississauga , 3359 Mississauga Road, Mississauga, Ontario L5L 1C6, Canada.|Impact Centre, University of Toronto , 112 College Street, Toronto, Ontario M5G 1A7, Canada.
authors.forename: David R
authors.identifier: 0000-0003-2676-5450
authors.initials: DR
authors.lastname: McMillen
mesh_terms: D002460:Cell Line; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014584:User-Computer Interface
chemical_list: D012330:RNA, Double-Stranded; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: RNA activation; double-stranded RNA; promoter; target prediction