At left, cells glow red to indicate that the detection system has been successfully delivered. The system was designed to produce green fluorescence in cells carrying a viral DNA sequence, as seen at right. Credit: Shimyn Slomovic
Biological engineers have developed a modular system of proteins that can detect a particular DNA sequence in a cell and then trigger a specific response, such as cell death. They needed to link these zinc fingers’ DNA-binding capability with a consequence – either turning on a fluorescent protein to reveal that the target DNA is present or generating another type of action inside the cell.
They used an”intein” – a short protein that can be inserted into a larger protein, splitting it into two pieces. The split protein pieces, known as “exteins,” only become functional once the intein removes itself while rejoining the two halves. Collins and Slomovic decided to divide an intein in 2 and then attach each portion to a split extein half and a zinc finger protein. The zinc finger proteins are engineered to recognize adjacent DNA sequences within the targeted gene, so if they both find their sequences, the inteins line up and are then cut out, allowing the extein halves to rejoin and form a functional protein. The extein protein is a transcription factor designed to turn on any gene the researchers want.
They linked green fluorescent protein (GFP) production to the zinc fingers’ recognition of a DNA sequence from an adenovirus, so that any cell infected with this virus would glow green. This reveals infected cells and also to kill them. To achieve this, they could program the system to produce proteins that alert immune cells to fight the infection, instead of GFP.
The MIT researchers also deployed this system to kill cells by linking detection of the DNA target to production of an enzyme called NTR. This enzyme activates a harmless drug precursor, CB 1954, which the researchers added to the petri dish where the cells were growing. When activated by NTR, CB 1954 kills the cells.
Future versions of the system could be designed to bind to DNA sequences found in cancerous genes and then produce transcription factors that would activate the cells’ own programmed cell death pathways.
The researchers are now adapting this system to detect latent HIV proviruses, which remain dormant in some infected cells even after treatment. Learning more about such viruses could help scientists find ways to permanently eliminate them. It could also be use it to test if genetic material has been successfully delivered to cells that scientists are trying to genetically alter. Cells that did not receive the new gene could be induced to undergo cell death, creating a pure population of the desired cells.
It could also be used to study chromosomal inversions and transpositions in cancer cells, or to study the 3-D structure of normal chromosomes by testing whether two genes located far from each other on a chromosome fold in such a way that they end up next to each other. http://news.mit.edu/2015/protein-sensor-detect-kill-cancer-cells-0921
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