Researchers at the University of Melbourne, Australia, have developed a DNA molecular switch that detects the internalization of materials in cells. The specific hybridization internalization probe (SHIP) designed by Haiyin Liu and Angus Johnston consists of two DNA sequences. One sequence – the fluorescence internalization probe (FIP) – has a Cy5 dye at the 5′ end and an azide at the 3′ end, which can be “clicked” onto the material that is being investigated. The other sequence – the quencher probe (QP) – can hybridize to the FIP and is functionalized with a Black Hole Quencher dye at the 3′ end.
The material of interest (protein or nanoparticle) is labeled with the FIP. Once the labeled material has been incubated with the cells for the appropriate period of time, the QP is added. It specifically quenches the fluorescence of the FIP on the surface by binding to FIP. However, the QP is not taken up by the cell and so it does not affect the fluorescence of internalized FIP. Flow cytometry is used to give a high-throughput analysis.
The advantages of this assay over existing techniques is that the DNA hybridization is very specific and the procedure can be used with a range of fluorescent probes. Furthermore, high concentrations and washing steps are not required. The researchers also confirmed that the labeling did not interfere with the internalization of the materials, and that the assay can be applied to nanoparticles, as these materials are frequently employed for drug delivery.
- A Programmable Sensor to Probe the Internalization of Proteins and Nanoparticles in Live Cells,
Haiyin Liu and Angus P. R. Johnston,
Angew. Chem. Int. Ed. 2013.