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Psoralen with a delivery peptide biotinylated


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Background

As an RNA polymerase tracks along helical DNA during transcription, positive supercoils of DNA are produced in front of the polymerase and negative supercoils are generated behind it (ref 1). These supercoils are then relaxed by topoisomerases. Matsumoto and Hirose developed a method to visualize the transcription-coupled negative supercoils in an interphase genome (ref 2). The technique relies on the ability of 4, 5’, 8-trimethyl psoralen (abbreviated as psoralen below) to intercalate into DNA. Upon exposure to 365 nm light, the intercalated psoralen crosslinks DNA strands at a rate that depends on the degree of negative superhelicity in the DNA. Using biotinylated psoralen and fluorescent streptavidine, the unconstrained negative supercoils can be visualized within a cell. The psoralen reagent was further improved by replacing a linker region between psoralen and biotin with a delivery peptide derived from HIV Tat protein (ref 3). This allows rapid incorporation of the reagent into cells without prior treatment with a detergent. The method is applicable to cultured cells, tissue slices and dissected out small tissues where transcription is going on.

Purity >95%
Chemical Formula

Application

Recommended use
[ Cultured cells ]

Exponentially growing culture was added a solution of the psoralen reagent to a final concentration of 0.2ng/ml and incubated for 10min under dark. The culture was then illuminated with a 365 nm lamp (UVL-21, UVP) for 10min under dark to crosslink the psoralen reagent. The cells were washed with phosphate-buffered saline (PBS) and fixed with ethanol. The biotinylated psoralen was detected with a fluorescent dye-conjugated streptavidine (e.g. Alexa Fluor 488-labeled streptavidine, Molecular Probes). Glass bottom culture dishes or chamber slides are convenient for the observation.

[ Tissue slices ]

Tissue was extirpated and cut into a slice of ~5mm square with ~1mm thick using a razor. The tissue slice was soaked in 0.2ml of a solution of the psoralen reagent (0.2ng/ml in PBS) in a depression slide for 10min under dark.  The slice was then illuminated with a 365 nm lamp as above. The slice was turned upside down after 5min and the illumination was continued for another 5min to assure even exposure to the light. After the light exposure, frozen sections were made from the tissue slice. The section was transferred onto a slide glass and fixed with ethanol. The biotinylated psoralen was detected as above.

[ Salivary glands of Drosophila ]

Salivary glands were dissected in a dissection buffer (10mM Hepes-KOH pH7.6, 5mM MgCl2, 5mM KCl, 130mM NaCl, 1% polyethylene glycol 6000) from third-instar larvae raised at 18°C. Four to five pairs of the gland were soaked in 40ul of a solution of the psoralen reagent (0.2ng/ml in dissection buffer) on a siliconized cover glass for 10 min under dark. The glands were then illuminated with a 365 nm lamp as above. After the light exposure, the glands were fixed with 40% acetic acid and squashed. The squashed samples were fixed with ethanol and the biotinylated psoralen was detected as above.


Strong signals of the psoralen reagent detected on the nuclei of human cancer cells

 

 

References

1. Liu, L.F. and Wang, J.C. Supercoiling of the DNA template during transcription. Proc. Natl. Acad. Sci. USA 84, 7024-7027, 1987
2. Matsumoto, K. and Hirose, S. Visualization of unconstrained negative supercoils of DNA on polytene chromosomes of Drosophila.
3. Wadia, J. S. and Dowdy, S. F. Protein transduction technology. Curr. Opin. Biotech. 12, 52-56, 2002

Product List

Product Name Cat# Quantity Price

Psoralen, Biotin DataSheet

CSR-NIG-L1-R1

3*4 NG ¥40,000
$534

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