title: Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference.
abstract: RNA interference (RNAi) is a natural cellular regulatory process that inhibits gene expression by transcriptional, post-transcriptional and translational mechanisms. Synthetic approaches that emulate this process (small interfering RNA (siRNA), short hairpin RNA (shRNA)) have been shown to be similarly effective in this regard. We developed a novel 'bifunctional' RNAi strategy, which further optimizes target gene knockdown outcome. A bifunctional construct (bi-sh-STMN1) was generated against Stathmin1, a critical tubulin modulator that is overexpressed in human cancers. The bifunctional construct is postulated to concurrently repress the translation of the target mRNA (cleavage-independent, mRNA sequestration and degradation) and degrade (through RNase H-like cleavage) post-transcriptional mRNA through cleavage-dependent activities. Bi-sh-STMN1 showed enhanced potency and durability in parallel comparisons with conventional shRNA and siRNAs targeting the same sequence. Enhanced STMN1 protein knockdown by bi-sh-STMN1 was accompanied by target site cleavage at the mRNA level showed by the rapid amplification of complementary DNA ends (RACE) assay. Bi-sh-STMN1 also showed knockdown kinetics at the mRNA level consistent with its multieffector silencing mechanisms. The bifunctional shRNA is a highly effective and advantageous approach mediating RNAi at concentrations significantly lower than conventional shRNA or siRNA. These results support further evaluations.
authors.affiliation: Gradalis, Inc., Dallas, TX, USA.
authors.forename: D D
authors.initials: DD
authors.lastname: Rao
authors.forename: P B
authors.initials: PB
authors.lastname: Maples
authors.forename: N
authors.initials: N
authors.lastname: Senzer
authors.forename: P
authors.initials: P
authors.lastname: Kumar
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.forename: B O
authors.initials: BO
authors.lastname: Pappen
authors.forename: Y
authors.initials: Y
authors.lastname: Yu
authors.forename: C
authors.initials: C
authors.lastname: Haddock
authors.forename: C
authors.initials: C
authors.lastname: Jay
authors.forename: A P
authors.initials: AP
authors.lastname: Phadke
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.forename: J
authors.initials: J
authors.lastname: Kuhn
authors.forename: D
authors.initials: D
authors.lastname: Dylewski
authors.forename: S
authors.initials: S
authors.lastname: Scott
authors.forename: D
authors.initials: D
authors.lastname: Monsma
authors.forename: C
authors.initials: C
authors.lastname: Webb
authors.forename: A
authors.initials: A
authors.lastname: Tong
authors.forename: D
authors.initials: D
authors.lastname: Shanahan
authors.forename: J
authors.initials: J
authors.lastname: Nemunaitis
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D050777:Stathmin / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; C493995:STMN1 protein, human; D050777:Stathmin