title: A random shRNA-encoding library for phenotypic selection and hit-optimization.
abstract: RNA interference (RNAi) is a mechanism for inhibiting gene expression through the action of small, non-coding RNAs. Most existing RNAi libraries target single genes through canonical pathways. Endogenous microRNAs (miRNAs), however, often target multiple genes and can act through non-canonical pathways, including pathways that activate gene expression. To interrogate all possible functions, we designed, synthesized, and validated the first shRNA-encoding library that is completely random at the nucleotide level. Screening in an IL3-dependent cell line, FL5.12, yielded shRNA-encoding sequences that double cell survival upon IL3 withdrawal. Using random mutagenesis and re-screening under more stringent IL3-starvation conditions, we hit-optimized one of the sequences; a specific nucleotide change and the creation of a mismatch between the two halves of the stem both contributed to the improved potency. Our library allows unbiased selection and optimization of shRNA-encoding sequences that confer phenotypes of interest, and could be used for the development of therapeutics and tools in many fields of biology.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Yongping
authors.initials: Y
authors.lastname: Wang
authors.forename: Yun E
authors.initials: YE
authors.lastname: Wang
authors.forename: M Grazia
authors.initials: MG
authors.lastname: Cotticelli
authors.forename: Robert B
authors.initials: RB
authors.lastname: Wilson
mesh_terms: D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D015723:Gene Library; D005821:Genetic Techniques; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D016296:Mutagenesis; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012190:Retroviridae / Q000378:metabolism
chemical_list: D007377:Interleukin-3; D035683:MicroRNAs; D034741:RNA, Small Interfering; D053148:Caspase 3
references.citation: Nat Genet. 2005 Nov;37(11):1281-8
references.pmid: 16200065
references.citation: Nature. 2004 Mar 25;428(6981):431-7
references.pmid: 15042092
references.citation: Nat Biotechnol. 2004 Mar;22(3):326-30
references.pmid: 14758366
references.citation: Nucleic Acids Res. 2004 Feb 09;32(3):893-901
references.pmid: 14769947
references.citation: FEBS Lett. 2005 Oct 31;579(26):5822-9
references.pmid: 16214139
references.citation: Nat Struct Mol Biol. 2004 Jul;11(7):599-606
references.pmid: 15170178
references.citation: Genes Dev. 2004 May 1;18(9):975-80
references.pmid: 15105377
references.citation: Nat Genet. 2004 Feb;36(2):183-9
references.pmid: 14704668
references.citation: Cell. 2005 Jan 14;120(1):15-20
references.pmid: 15652477
references.citation: Nature. 2004 Mar 25;428(6981):427-31
references.pmid: 15042091
references.citation: Nat Chem Biol. 2007 Mar;3(3):166-73
references.pmid: 17259978
references.citation: Mol Cell. 1999 Feb;3(2):159-67
references.pmid: 10078199
references.citation: Methods Enzymol. 1987;155:558-68
references.pmid: 3481016
references.citation: Nature. 2005 Feb 17;433(7027):769-73
references.pmid: 15685193
references.citation: Dev Biol. 2002 Mar 15;243(2):215-25
references.pmid: 11884032
references.citation: RNA. 2004 Mar;10(3):387-94
references.pmid: 14970384
references.citation: Proc Natl Acad Sci U S A. 2004 Apr 13;101(15):5494-9
references.pmid: 15024103
references.citation: Nature. 2007 Dec 13;450(7172):1117-20
references.pmid: 18075597
references.citation: Proc Natl Acad Sci U S A. 2006 Nov 14;103(46):17337-42
references.pmid: 17085592
references.citation: RNA. 2006 Jun;12(6):988-93
references.pmid: 16611941
references.citation: Nat Biotechnol. 2005 Feb;23(2):227-31
references.pmid: 15619616
references.citation: Dev Biol. 1999 Dec 15;216(2):671-80
references.pmid: 10642801
references.citation: Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2356-61
references.pmid: 15695593
references.citation: Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6047-52
references.pmid: 11972060
references.citation: Nature. 2004 Dec 23;432(7020):1036-40
references.pmid: 15616564
references.citation: Science. 2007 Dec 21;318(5858):1931-4
references.pmid: 18048652
references.citation: Nat Genet. 2004 Feb;36(2):190-6
references.pmid: 14704669
references.citation: Nature. 2003 Aug 14;424(6950):797-801
references.pmid: 12917690
references.citation: Nat Biotechnol. 2005 Feb;23(2):222-6
references.pmid: 15619617
references.citation: J Pharm Sci. 2007 Nov;96(11):2922-30
references.pmid: 17518360
references.citation: Nature. 2007 Jul 5;448(7149):39-43
references.pmid: 17572664