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With $4M from NIH, Chaput, LaBaer Building High-Throughput Affinity Agent Production Pipeline


By Adam Bonislawski

Researchers at Arizona State University's Biodesign Institute have received a three-year, $4 million grant from the National Institutes of Health to develop a high-throughput pipeline for generating synthetic affinity reagents called DNA synbodies.

Consisting of pairs of peptides linked by a DNA backbone, these reagents are significantly easier and cheaper to produce than conventional affinity reagents like antibodies and, in early studies, have shown equivalent or better sensitivity and specificity.

In August, a team of ASU researchers led by John Chaput, one of the inventors of the technology, published a paper in ChemBioChem demonstrating the effectiveness of DNA synbodies targeting the cancer marker Grb2 in HeLa cell lysate (PM 8/19/2011).

Now, Chaput said, his team plans to use to the NIH funds to automate the synbody production and selection process, working in collaboration with the lab of Joshua LaBaer, the Biodesign Institute's chair in personalized medicine.

"We're going to try to put together a pipeline that, when we're done, will be completely automated using robotics technology," Chaput told ProteoMonitor. The scientists, he noted, have previously performed each step of the process individually, "so now it's kind of just a matter of putting it together and packaging it all up."

"We anticipate we'll have the pipeline up and running in about the first 12 months, and then from that point forward there will be a lot of optimization at various stages," he said. "We'll also spend a lot of time characterizing the reagents coming out of the pipeline."

The anti-Grb2 synbodies used in the ChemBioChem study were constructed via literature searches through which the researchers identified peptides known to bind to the protein. The high-throughput platform, on the other hand, will use mRNA display technology to create large combinatorial libraries containing as many as 1012 peptide sequences against which proteins can be screened.

To produce the synbodies, peptide pairs are attached to a DNA backbone in a variety of configurations via standard amine coupling chemistry. The synbodies are then screened against target proteins using surface plasmon resonance to determine which peptide-backbone configuration offers the optimal binding.

"The design of the reagents is pretty straightforward when it comes to assembling them on a larger platform," Chaput said. "For example, we can do our peptide discovery process in a 96-well format. We can build them on individual strands of DNA also in a 96-well format. And then we have bivalent affinity reagents that we can screen against individual targets at that point and see which combination is best in terms of its distance and angular geometry."

LaBaer's lab will bring its automation expertise to the project, Chaput said. Perhaps more important, it will bring its tools for making the protein targets that potential synbodies can be screened against. LaBaer runs the Biodesign Institute's DNASU plasmid repository, which contains more than 147,000 plasmids and last week received a separate $6.5 million grant from NIH. That resource and LaBaer's research into high-throughput protein production are key to the synbody project, Chaput said.

"The [DNASU] repository is one of the major repositories of genes in the country, and so these are going to be the genes we use to create the proteins that will be used to generate these affinity reagents," he said.

"The call from NIH is to develop a high-throughput way of building these sorts of reagents," LaBaer told ProteoMonitor. "They're not just interested in having outstanding reagents; they're interested in eventually scaling this up to the proteome."

"Obviously, if you're going to be selecting synbodies, you need proteins to select them against," he said. "So we've spent some time developing high-throughput protocols for expressing and purifying proteins in quantities that we think are going to be adequate to make these synbodies."

LaBaer is one of the inventors of the nucleic acid programmable protein array, or NAPPA – a protein production platform in which proteins are synthesized in situ directly from printed cDNA vectors (PM 5/15/2008).

While antibody production can require milligrams of protein, synbody screening needs only microgram levels, making it compatible with such high-throughput protein production methods, LaBaer said.

The researchers plan to focus on kinases to start, with their aim being to generate 40 candidate synbodies to each of 100 different kinases by the end of the three-year funding period.

"To a certain extent that is a conservative goal, because it's quite possible that we can generate affinity agents to a lot more than 100 proteins when it's all said and done," Chaput said. "But because this is also a technology-development grant, we said, 'Let's really take our time and work out all the chemistry and biology at each step.'"

Thorough characterization of the synbodies is a primary goal, he added.

"This is one of the shortfalls of antibodies – you just don't get a lot of biophysical information when you buy an antibody from a commercial company," he said. "So for a subset of the reagents we're going to do a thorough characterization. We'll know their kinetic rates, their on-rate of binding, their off-rate of binding, their affinities, their specificities."

To this end they will be aided by a prototype surface plasmon resonance machine that LaBaer recently obtained for his lab from SPR firm Plexera Bioscience. Unlike most SPR instruments, the prototype is compatible with NAPPA technology, "so we can produce any protein on an array using NAPPA, stick it on the SPR device, and measure it using that," LaBaer said.

Chaput said he anticipates his team will be able to generate synbodies with binding affinities in the 50- to 100-picomolar range, "which would be at the binding affinity range of the very best antibodies that are out there." Making a novel synbody takes several days, he said, compared to months for an antibody.

The initial version of the pipeline will use proteins produced in E. coli, which "is the easiest system to get up and running," Chaput said. "Eventually we may shift over to a mammalian system to be able to target proteins with post-translational modifications."

Chaput's team has submitted a patent covering the reagents and is currently in discussion with the Biodesign Institute's Impact Accelerator program, which works to move scientific discoveries into the marketplace. No decisions have been made yet regarding a commercialization strategy for the technology, he said, but the researchers have seen "some interest from the outside community" since the release of the ChemBioChem paper.

Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.