Golden touch with zinc fingers

MIT researchers use zinc fingers to detect DNA sequence and then pull trigger on cancer cells

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CAMBRIDGE, Mass.—Armed with new weapons aimed at killing cancer cells and cells infected with a virus, biological engineers at the Massachusetts Institute of Technology (MIT) have developed a modular system of proteins targeted toward detecting a particular DNA sequence in a cell—then triggering a cell death. The technology, described in the Sept. 21 scientific journal Nature Methods, is based on DNA-binding proteins known as zinc fingers.
“There is a range of applications for which this could be important,” says James Collins, senior author and Termeer Professor of Medical Engineering and Science in MIT’s Department of Biological Engineering and Institute of Medical Engineering and Science (IMES). “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.”
Shimyn Slomovic, a postdoctoral associate of IMES and lead author on the journal paper about targeting DNA, has said, “The technologies are out there to engineer proteins to bind to virtually any DNA sequence. We felt that there was a lot of potential in harnessing this designable DNA-binding technology for detection.”
Cancer generally works through mutated DNA sequences that are either passed on from previous generations or acquired throughout life and environmental exposure. Therefore, it makes sense that detecting specific DNA mutations that lead to cancer can play a role in combating this disease, Slomovic tells DDNews.
“Today, technologies exist that allow researchers and clinicians to read DNA sequences purified from patient cells and discover mutations,” Slomovic explains. “And in parallel, we are getting better at designing proteins to recognize specific sequences, such as those used in our study.”
One benefit in using designable proteins for DNA recognition “lies in the ability to interrogate the DNA inside living cells,” she says. “Although there are hurdles to overcome, such protein devices could someday play a direct role in cancer diagnosis and/or treatment and now may be used as research tools that expand our knowledge of the inner workings of cancer.”
This platform has the potential to lead to several different avenues, one of which could be the detection of DNA mutations or chromosomal defects that lead to cancer, she notes.
“Presently, we are gearing toward the use of this system to aid in identifying and eradicating the HIV provirus, whose DNA can enter a dormant state after it has embedded itself within the human genome,” Slomovic explains. “This latency allows the virus to evade antiviral treatments that are administered to infected individuals—and its eradication may be the final stage in facilitating a cure to the disease.”
An additional direction might be to expand the detection repertoire of the current system to include pathogens whose genomes exist as RNA, such as the Ebola virus or SARS.
To create their new system, researchers needed to first link 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 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 approach could be used not only to reveal infected cells, but also to kill them,” Slomovic notes. “To achieve this, the researchers could program the system to produce proteins that alert immune cells to fight the infection, instead of GFP.”
“Since this is modular, you can potentially evoke any response that you want,” Slomovic says. “You could program the cell to kill itself, or to secrete proteins that would allow the immune system to identify it as an enemy cell so the immune system would take care of it.”
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.
Adds Collins: “While treating diseases using this system is likely many years away, it could be used much sooner as a research tool. For example, scientists could use it to test whether genetic material has been 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.

MIT-engineered viruses could combat human disease, improve food safety
CAMBRIDGE, Mass—In the war against harmful bacteria, scientists have turned to a familiar opponent: viruses that infect bacteria. By tweaking the genomes of these viruses, known as bacteriophages, researchers hope to customize them to target any type of pathogenic bacteria.
To do this, MIT biological engineers have “devised a new mix-and-match system to genetically engineer viruses that target specific bacteria. This approach could generate new weapons against bacteria for which there are no effective antibiotics,” says Timothy Lu, an associate professor of electrical engineering and computer science and biological engineering. Lu is the senior author of a paper describing this work in the Sept. 23 edition of the journal Cell Systems.
These modular bacteriophages “could also be used to ‘edit’ microbial communities, such as the population of bacteria living in the human gut, Lu said. “There are trillions of bacterial cells in the human digestive tract, and while many of these are beneficial, some can cause disease.”
Some reports have linked Crohn’s disease to the presence of certain strains of E. coli, he added.
“We’d like to be able to remove specific members of the bacterial population and see what their function is in the microbiome,” Lu said. “In the longer term you could design a specific phage that kills that bug but doesn’t kill the other ones—but more information is needed to effectively design such therapies.”
The Food and Drug Administration (FDA) has approved a handful of bacteriophages for treating food products, but efforts to harness them for medical use have been hampered because isolating useful phages from soil or sewage can be a tedious, time-consuming process,” Lu notes.
The journal article explains that after the researchers identified the genes to insert into their phage scaffold, they created a new system for performing the genetic engineering. Existing techniques for editing viral genomes are fairly laborious, so the researchers came up with an efficient approach in which they insert the phage genome into a yeast cell, where it exists as an “artificial chromosome” separate from the yeast cell’s own genome.
 “Once we had that method, it allowed us very easily to identify the genes that code for the tails and engineer them or swap them in and out from other phages,” Lu said.

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