Light controllable siRNAs regulate gene suppression and phenotypes in cells.
Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs during transfection, limiting its use is in kinetic studies, deciphering toxic and off-target effects and phenotypic assays requiring temporal, and/or spatial regulation. We developed a novel controllable siRNA (csiRNA) that is activated by light. A single photo removable group is coupled during oligonucleotide synthesis to the 5' end of the antisense strand of the siRNA, which blocks the siRNA's activity. A low dose of light activates the siRNA, independent of transfection resulting in knock down of specific target mRNAs and proteins (GAPDH, p53, survivin, hNuf2) without stimulating non-specific effects such as regulated protein kinase PKR and induction of the interferon response. We demonstrate survivin and hNuf2 csiRNAs temporally knockdown their mRNAs causing multinucleation and cell death by mitotic arrest, respectively. Furthermore, we demonstrate a dose-dependent light regulation of hNuf2 csiRNA activity and resulting phenotype. The light controllable siRNAs are introduced into cells using commercially available reagents including the MPG peptide based delivery system. The csiRNAs are comparable to standard siRNAs in their transfection efficiency and potency of gene silencing. This technology should be of interest for phenotypic assays such as cell survival, cell cycle regulation, and cell development.
Nguyen
Quan N
QN
Genospectra, Inc., 6519 Dumbarton Circle, Fremont, CA 94555, USA.
Chavli
Rajesh V
RV
Marques
Joao T
JT
Conrad
Peter G
PG
Wang
Die
D
He
Weihai
W
Belisle
Barbara E
BE
Zhang
Aiguo
A
Pastor
Larry M
LM
Witney
Frank R
FR
Morris
May
M
Heitz
Frederic
F
Divita
Gilles
G
Williams
Bryan R G
BR
McMaster
Gary K
GK
D001681:Biological Assay; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D004268:DNA-Binding Proteins / Q000008:administration & dosage; D015870:Gene Expression / Q000187:drug effects*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008027:Light; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D010641:Phenotype; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000737:chemistry* / Q000528:radiation effects*; D000077022:Survivin; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics
C107489:BIRC5 protein, human; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051034:Inhibitor of Apoptosis Proteins; C475074:MPG peptide; D008869:Microtubule-Associated Proteins; C490197:NUF2 protein, human; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D000077022:Survivin; D016159:Tumor Suppressor Protein p53; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases