title: An optimized microRNA backbone for effective single-copy RNAi.
abstract: Short hairpin RNA (shRNA) technology enables stable and regulated gene repression. For establishing experimentally versatile RNAi tools and minimizing toxicities, synthetic shRNAs can be embedded into endogenous microRNA contexts. However, due to our incomplete understanding of microRNA biogenesis, such "shRNAmirs" often fail to trigger potent knockdown, especially when expressed from a single genomic copy. Following recent advances in design of synthetic shRNAmir stems, here we take a systematic approach to optimize the experimental miR-30 backbone. Among several favorable features, we identify a conserved element 3' of the basal stem as critically required for optimal shRNAmir processing and implement it in an optimized backbone termed "miR-E", which strongly increases mature shRNA levels and knockdown efficacy. Existing miR-30 reagents can be easily converted to miR-E, and its combination with up-to-date design rules establishes a validated and accessible platform for generating effective single-copy shRNA libraries that will facilitate the functional annotation of the genome.
authors: Mirimus Inc., 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA. Electronic address: fellmann@mirimus.com.
authors: Christof
authors: C
authors: Fellmann
authors: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
authors: Thomas
authors: T
authors: Hoffmann
authors: Mirimus Inc., 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
authors: Vaishali
authors: V
authors: Sridhar
authors: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
authors: Barbara
authors: B
authors: Hopfgartner
authors: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
authors: Matthias
authors: M
authors: Muhar
authors: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
authors: Mareike
authors: M
authors: Roth
authors: Mirimus Inc., 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
authors: Dan Yu
authors: DY
authors: Lai
authors: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
authors: Inês A M
authors: IA
authors: Barbosa
authors: Mirimus Inc., 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
authors: Jung Shick
authors: JS
authors: Kwon
authors: Mirimus Inc., 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
authors: Yuanzhe
authors: Y
authors: Guan
authors: Mirimus Inc., 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
authors: Nishi
authors: N
authors: Sinha
authors: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria. Electronic address: zuber@imp.ac.at.
authors: Johannes
authors: J
authors: Zuber
mesh_terms: D045744:Cell Line, Tumor; D055785:Gene Knockdown Techniques / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D059372:Nucleotide Motifs
chemical_list: C546086:MIRN30b microRNA, human; D035683:MicroRNAs