Remote Gene Switch
To study the role of a certain gene (and its product) in the life of cells, tissues or the whole organism, there are several techniques. For example, you can "turn off" a gene, reduce its activity to a minimum – and see what changes will occur in the cell.
There are several ways to inactivate a gene. One of them, very elegant, promising and widely used by scientists at the present time, was developed by nature itself. It turns out that in eukaryotic cells there is a special multicomponent complex capable of selectively directing information RNA (mRNA) to degradation. Recall that mRNA is one of the stages on the way from a gene to a protein. If there is no mRNA, there is no protein, that is, as if there is no gene activity. The selectivity of degradation is due to the presence of an oligonucleotide sequence in the complex, complementary to the target mRNA. It turned out that oligonucleotides artificially introduced into cells are also able to turn on the natural mechanism of mRNA degradation.
Researchers from America have proposed a way to control the "shutdown" of genes at the desired time. To do this, the following construction was created (Figure 1).
Figure 1. The principle of operation of the "switches": after irradiation with near infrared light, the gold rods are heated, and the double helix of the nucleic acid is destroyed.
A single chain of a double-stranded nucleic acid molecule was covalently sewn to the gold nanorode. The size of the rod and the ratio of length to diameter equal to 3.5 were chosen so that the particle had maximum absorption when irradiated with near infrared light (785 nm). When irradiated, the particle heats up, the complementary bonds between the oligonucleotides are destroyed, and the released single-stranded nucleic acid molecules can participate in the degradation of mRNA.
As is customary, the researchers first showed that the proposed system works in vitro. The gold particles were immobilized on a glass substrate. To visualize the process, a fluorescent label was sewn to the second (unrelated to the nanoparticle) DNA chain. After irradiation and melting of DNA, labeled oligonucleotides were released from the complex with the gold particle, as expected (Figure 2).
Figure 2. Change in the fluorescence of nanoparticles after irradiation with light (785 nm) in vitro.
For in vivo experiments, the researchers selected human breast carcinoma cells with ErbB2 receptors on the surface. It was decided to "turn off" the synthesis of these receptors in a demonstration. The choice seems somewhat strange to an inexperienced observer – primarily because these receptors are able to exist for quite a long time. And since there is no way to distinguish between newly synthesized and previously created receptors, it becomes quite difficult to notice a stop in the synthesis of this protein. The authors themselves say that some effect becomes noticeable only 48 hours after irradiation (Figure 3).
Figure 3. The number of ErbB2 receptors in human breast carcinoma cells (flow cytometry method). (A) Control: non-activated "switches", without irradiation. (C) Control: cells are irradiated with infrared light, but do not contain nanoparticles.(C) Control: irradiation-activated nanoparticles containing an unsuitable oligonucleotide sequence. (D) Experience: activated "switches" for ErbB2 receptors. Cells with a reduced number of receptors are marked in blue.
The developed method allows targeted control of gene activity in individual cells. There is no doubt that the work is attractive both for fundamental research and in the therapeutic aspect. By the way, perhaps it is the authors' focus on "practice" that explains the choice of cells and protein targets for experiments.
The work "Remote Optical Switch for Localized and Selective Control of Gene Interference" is published in Nano Letters.