Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference.
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.
Rao
D D
DD
Gradalis, Inc., Dallas, TX, USA.
Maples
P B
PB
Senzer
N
N
Kumar
P
P
Wang
Z
Z
Pappen
B O
BO
Yu
Y
Y
Haddock
C
C
Jay
C
C
Phadke
A P
AP
Chen
S
S
Kuhn
J
J
Dylewski
D
D
Scott
S
S
Monsma
D
D
Webb
C
C
Tong
A
A
Shanahan
D
D
Nemunaitis
J
J
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
D012333:RNA, Messenger; D034741:RNA, Small Interfering; C493995:STMN1 protein, human; D050777:Stathmin