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Results: 7

1.
Scheme 3.

Scheme 3. From: Optochemical control of RNA interference in mammalian cells.

Synthesis of the NPOM-caged uridine phosphoramidite 13 from 8. Dimethylformamide (DMF), tert-butyldimethylsilyl chloride (TBDMSCl), dichloromethane (DCM), di(p-methoxyphenyl)phenyl-methyl chloride (DMTCl), tetrahydrofuran (THF) and N,N-diisopropylethylamine (DIPEA).

Jeane M. Govan, et al. Nucleic Acids Res. 2013 December;41(22):10518-10528.
2.
Scheme 2.

Scheme 2. From: Optochemical control of RNA interference in mammalian cells.

Synthesis of the NPOM-caged guanosine phosphoramidite 7 from commercially available 1. Dimethylformamide (DMF), tert-butyldimethylsilyl chloride (TBDMSCl), dichloromethane (DCM), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), di(p-methoxyphenyl)phenyl-methyl chloride (DMTCl) and tetrahydrofuran (THF).

Jeane M. Govan, et al. Nucleic Acids Res. 2013 December;41(22):10518-10528.
3.
Figure 1.

Figure 1. From: Optochemical control of RNA interference in mammalian cells.

Sequences and Tm of caged siRNAs. Bold and underLined G denotes a caged guanosine nucleotide from the incorporation of 7 and a bold and underLined U denotes a caged uridine nucleotide from the incorporation of 13. Standard deviations were calculated form three individual experiments.

Jeane M. Govan, et al. Nucleic Acids Res. 2013 December;41(22):10518-10528.
4.
Scheme 1.

Scheme 1. From: Optochemical control of RNA interference in mammalian cells.

Two siRNA light-activation approaches. Caged nucleotides are positioned (A) near the cleavage site or (B) at the seed region of the siRNA agent, leading to gene expression by preventing RISC cleavage or mRNA target recognition. On UV irradiation, the caging groups are cleaved resulting in the silencing of gene expression through RNA interference.

Jeane M. Govan, et al. Nucleic Acids Res. 2013 December;41(22):10518-10528.
5.
Figure 4.

Figure 4. From: Optochemical control of RNA interference in mammalian cells.

Quantification of photoactivation of Eg5 siRNA. HeLa cells were transfected with siRNAs, and after 48-h incubation, the RNA was extracted and quantitative real-time PCR analysis was performed. Eg5 expression was normalized to the expression of the GAPDH housekeeping gene, and the negative control was set to 100% Eg5 expression. Error bars represent standard deviations from three independent experiments.

Jeane M. Govan, et al. Nucleic Acids Res. 2013 December;41(22):10518-10528.
6.
Figure 2.

Figure 2. From: Optochemical control of RNA interference in mammalian cells.

Photoactivated RNA interference in mammalian cells. HEK 293T cells were transfected with pEGFP-N1, pDsRed-N1 monomer and siRNA oligonucleotides. Cells were irradiated for 5 min (25 W, 365 nm) or kept in the dark. (A–F) Cells were imaged after 48 h. The GFP channel is shown above the DsRed channel. (G) After a 48-h incubation, the cells were trypsinized and analyzed by flow cytometry. The number of cells expressing both GFP and DsRed was normalized to the number of cells expressing only DsRed. Standard deviations were calculated from three individual experiments.

Jeane M. Govan, et al. Nucleic Acids Res. 2013 December;41(22):10518-10528.
7.
Figure 3.

Figure 3. From: Optochemical control of RNA interference in mammalian cells.

Optochemical activation of Eg5 siRNA in HeLa cells. HeLa cells were transfected with caged and non-caged siRNAs (40 pmol). The cells were irradiated (5 min, 25 W, 365 nm) and incubated at 37°C, 5% CO2 for 48 h. (A–G) The cells were fixed and stained with Alexa Fluor 488 phalloidin (green) and DAPI (blue). The cells were imaged on a Zeiss LSM 710 confocal microscope using a 40× oil objective and Alexa Fluor 488 and DAPI-specific lasers (488 nm multiline argon and 405 nm diode).

Jeane M. Govan, et al. Nucleic Acids Res. 2013 December;41(22):10518-10528.

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