Researchers at Harvard and London-based Gendaq have used microarrays to detect DNA binding sites for zinc finger proteins, key molecules involved in gene regulation.
This effort, which the researchers and colleagues at the UK’s Medical Research Council described in an article published June 12 in the Proceedings of the National Academy of Sciences, was aimed at characterizing lower affinity DNA zinc finger interactions, as well as the effect of mutations in DNA binding sites on the DNA zinc finger interactions.
They said they chose microarray technology because it allows a number of interactions to be measured at once, picks up weak as well as strong interactions, and can be done more cost effectively.
“It comes down to being able to do things in a very highly parallel way,” said Yen Choo, Gendaq’s chief scientific officer and an author of the paper. “It gives you information on all the different binding sites whereas [other techniques] give you only the winners — those that bind most tightly” to the zinc finger.
Microarray assays could also be done more quickly and cheaply than conventional techniques for detecting zinc finger binding sites such as gel mobility-shift assays and nitrocellulose-binding assays, the researchers wrote in their paper.
In their work, the researchers spotted onto glass slides double-stranded DNA oligonucleotides, which they selected from numerous possible binding sites for a specific zinc finger protein, the mouse transcription factor Zif268. They chose this transcription factor as a means to test the microarray technology because its interaction with DNA has already been well characterized through crystallography, but specific information about the sequence to which it binds preferentially had not been previously elucidated.
Using this method, researchers were able to determine the sequences to which the transcription factors preferentially bind. They were also able to learn about the binding patterns of mutant transcription factors.
Gendaq sought to detect the zinc finger-DNA interactions as part of its efforts to develop the Z-switch, its marquis product for gene regulation experiments. Z-switches are zinc finger proteins specially engineered in a lab to force particular genes in a eukaryotic cell to be up- or down-regulated. Researchers can use them to study the effects of particular genes on cellular functions in vivo, without disturbing the development of the cell.
These microarrays “allow us to be able to quality control the Z-switches we produce,” said Choo. “We used to use a similar technique on microtiter plates, but obviously, you would have to use a lot of plates to get a large number of samples done simultaneously.”
The authors also suggested microarray technology could be used to study other gene promoters against transcription factors or to perform whole-genome analyses of transcription factor binding sites.
“Especially in sequenced organisms like yeast, you can array all those areas of the genome you suspect to be important in gene regulation, then you can ask the question: to which of these regions does a particular transcription factor bind?” Choo said.
Gendaq, which Sangamo Biosciences recently obtained the option to acquire in a stock deal worth $39.7 million, is a privately held offshoot of the Medical Research Council’s Laboratory of Molecular Biology in Cambridge, UK. The company was assigned the intellectual property for the lab’s zinc finger technology, and has been commercializing this technology as the Z-switch.
In addition to the June 12 paper, Gendaq has published two previous papers in PNAS that describe the science behind its technology. The two papers, which appeared in February, outlined new methods of constructing improved zinc finger protein multimers, biomolecules that bind to DNA sequences with high specificity. Gendaq uses these multimers to make Z-switches.