Light controllable siRNAs regulate gene suppression and phenotypes in cells

Genospectra, Inc., 6519 Dumbarton Circle, Fremont, CA 94555, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 04/2006; 1758(3):394-403. DOI: 10.1016/j.bbamem.2006.01.003
Source: PubMed

ABSTRACT 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.

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Available from: Die Wang, Oct 14, 2014
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    • "The uncaging technique proved to be extremely successful for observing fast biological processes by their rapid manipulation in time (Kaplan and Somlyo, 1989; McCray et al., 1980; McCray and Trentham, 1989) and it is still a useful and popular in vitro method for rapid enzyme kinetic studies (Artamonov et al., 2013). The method enables us to manipulate the active concentrations of small molecules, peptides, or nucleic acids (Mayer and Heckel, 2006; Nguyen et al., 2006), track dynamic processes in vivo, or enhance imaging by applying photo-caged fluorescent compounds (Guo et al., 2008). In the process of uncaging , a protecting group is photolyzed by a UV flash and the bioactive compound is released. "
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    • "The choice of inducer molecules is also inherently limited by the availability of the transcription factors. In another approach, several groups developed chemically modified siRNA derivatives that are activated by light [7] [8] [9] [10]. These photocaged siRNAs are induced irreversibly and often to suboptimal levels. "
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    • "It can be said that these two sequences worked best considering inactivation as well as activation capability. In accord with another group's observation (Nguyen et al. 2006), in some cases we found that impurities of already deprotected sequences (identified by RP-HPLC) in a range of z3% can cause eminent loss of inactivation. A more important aspect is the time up to which the caged sequences remain inactive inside cells; 28 h after transfection, a reactivation of the siRNA sets in even without irradiation. "
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