A thumbs-up with zinc fingers

CAMBRIDGE, Mass.—Zinc finger proteins, a type of DNA-binding protein, play a variety of roles within the human body, and an MIT team is hoping to take advantage of that versatility in potential research applications. Biological engineers at MIT used these proteins to develop a modular system capable of detecting particular DNA sequences and triggering cell responses, and published their results in Nature Methods. The work was led by James Collins, the Termeer Professor of Medical Engineering and Science in MIT’s Department of Biological Engineering and Institute of Medical Engineering and Science (IMES), and Shimyn Slomovic, an IMES postdoc. Collins is the senior author of the paper, and Slomovic is the lead author.

While zinc fingers can recognize any protein, the team needed to combine them with a particular consequence, such as turning on a fluorescent protein to show the target DNA is present or generating some other reaction within a cell.

They decided to go with another protein known as an “intein,” a short protein that can be inserted into a larger protein to split it in two pieces. Those pieces, referred to as “exteins,” are only functional once the intein removes itself while rejoining the two halves.

The researchers divided an intein, then attached each portion to a split extein half and a zinc finger protein. Since the zinc finger proteins are designed to recognize adjacent DNA sequences within the targeted gene, if they both find their proper sequences, the inteins line up and are cut out, enabling the extein halves to rejoin and form a functional protein. For its part, the extein protein is a transcription factor designed to be capable of turning on any gene.

“There is a range of applications for which this could be important,” commented Collins. “This allows you to readily design constructs that enable a programmed cell to both detect DNA and act on that detection, with a report system and/or a respond system.”

For this work, green fluorescent protein (GFP) production was linked to the zinc fingers' recognition of a DNA sequence from an adenovirus, in order to make any cell containing the virus glow green. The benefit is that not only could this approach reveal infected cells, it could also kill them if the system is programmed to produce proteins that alert immune cells to fight the infection, rather than producing GFP.

This system also proved capable of killing cells when detection of the DNA target was linked to the production of an enzyme known as NTR, which activates the harmless drug precursor CB 1954 that, when activated by NTR, kills the target cells.

The team is now interested in adapting the system to detect latent HIV proviruses, which can remain dormant in infected cells even after treatment. Though Collins noted that using this system as a treatment is many years off, he pointed to its potential as a research tool, helping scientists determine whether genetic material has successfully reached target cells. The team also pointed out that this approach could also be used to study the chromosomal inversions and transpositions that take place in cancer cells or the 3D structure of normal chromosomes by investigating whether two genes located separately on a chromosome will end up next to each other once the chromosome is folded.

“The technologies are out there to engineer proteins to bind to virtually any DNA sequence that you want,” said Slomovic. We felt that there was a lot of potential in harnessing this designable DNA-binding technology for detection.”

SOURCE: MIT press release