A fluorescent image of the cell taken four hours into the same experiment. At this time the quantum dot-siRNA complex is distributed throughout the cellular fluid. The dark region in the middle of the cell is the nucleus. Credit: University of Washington
New work helps to overcome a long standing barrier in the siRNA (silence/turn off genes using RNA) field: How to achieve high silencing efficiency with low toxicity. The new quantum dot approach is 5-10 times less toxic and 7 to 25 times more effective. (98% of gene activity was stopped).
Quantum dots, fluorescent balls of semiconductor material just six nanometers across, was surrounded by a proton sponge that carried a positive charge. Without any quantum dots attached, the siRNA’s negative charge would prevent it from penetrating a cell’s wall. With the quantum-dot chaperone, the more weakly charged siRNA complex crosses the cellular wall, escapes from the endosome (a fatty bubble that surrounds incoming material) and accumulates in the cellular fluid, where it can do its work disrupting protein manufacture.
Key to the newly published approach is that researchers can adjust the chemical makeup of the quantum dot’s proton-sponge coating, allowing the scientists to precisely control how tightly the dots attach to the siRNA.
Quantum dots were dramatically better than existing techniques at stopping gene activity. In experiments, a cell’s production of a test protein dropped to 2 percent when siRNA was delivered with quantum dots. By contrast, the test protein was produced at 13 percent to 51 percent of normal levels when the siRNA was delivered with one of three commercial reagents, or reaction-causing substances, now commonly used in laboratories.
Central to the finding is that fluorescent quantum dots allow scientists to watch the siRNA’s movements. Previous siRNA trackers gave off light for less than a minute, while quantum dots, developed for imaging, emit light for hours at a time. In the experiments the authors were able to watch the process for many hours to track the gene-silencer’s path.
The new approach is also five to 10 times less toxic to the cell than existing chemicals, meaning the quantum dot chaperones are less likely to harm cells.
They were able to target a particular type of cell of gene therapy treatment. They are working to generalize and identify DNA sequences for targeting other types of cells.