Scientists at Arizona State University's Biodesign Institute have developed a new form of affinity reagent that could offer an easy-to-produce alternative to traditional antibodies.
Dubbed DNA synbodies, the agents consist of pairs of peptides linked by a DNA backbone. In an early study they showed equivalent or better sensitivity and specificity compared to commercial antibodies, said John Chaput, an associate professor at ASU and one of the inventors of the technology.
Key to the technology, Chaput told ProteoMonitor, is the use of two peptides per reagent, each binding a different site on the target protein. While individually neither peptide may bind with sufficient sensitivity or specificity, combining them can improve their performance by several orders of magnitude.
"Most of the peptides on their own are not all that great," he said. "Usually they're low micromolar binders, which is sufficient to be used in some types of biochemical analyses or validation studies, but they're not suitable as an affinity reagent because they just don't bind tight enough.
"But by connecting them on DNA at the perfect distance and geometry, we could transform them into a nanomolar binder," he said. "So that's a big jump. That's about a thousand-fold improvement in affinity, so when you do this it's not just additive."
In research detailed in a paper published this week in ChemBioChem, Chaput and his team generated synbodies targeting the protein Grb2, which has been implicated in a variety of diseases including breast, bladder, and prostate cancer, and compared their performance to that of two commercial mouse monoclonal anti-Grb2 antibodies — one from Santa Cruz Biotechnology and one from Thermo Scientific.
The researchers compared the three reagents' ability to detect Grb2 spiked into HeLa cell lysate, finding that the DNA synbody demonstrated a five-fold to ten-fold improvement in sensitivity compared to the commercial antibodies and equivalent specificity, and suggesting, Chaput said, that the technology has the potential to compete with traditional affinity agents.
The crucial advantage of synbodies compared to conventional antibodies, he noted, is the ease with which they can be constructed. Antibodies can take months to produce, while, according to Chaput, producing a novel synbody takes just several days.
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 using surface plasmon resonance to determine which peptide-backbone configuration offers the optimal binding.
"It's a pretty powerful technology that's chemically pretty simple," Chaput said. "We're not infecting mice; we're not infecting animals; we're not working with complex cell lines; and we're not performing iterative rounds of in vitro selection and amplification. We're just taking peptides and putting them on DNA and quickly identifying the precise distances and geometries that are required to transform these things to very high-affinity binders."
For the Grb2 work, the researchers selected the peptides to be used as binders via a literature search — a strategy, Chaput said, that will likely be effective for well-studied proteins like common disease biomarkers.
"In many cases there are either NMR structures or x-ray crystallography structures that have been solved with ligands bound to these proteins," he said. "So it just provides a huge amount of information to work with."
More complicated is identifying peptides for binding proteins that are less well characterized. Initially, the researchers investigated using peptide microarrays for this sort of work, screening target proteins against collections of several thousand peptides to identify good potential binders. While they were able in many situations to identify micromolar-level binders, they had less success subsequently optimizing these peptides, Chaput said.
"One of the questions was [whether you] can take [a peptide] that you identify off a microarray and optimize it very quickly by directed evolution," he said. "And it turns out that they're just not very evolvable; they're just not very easy to optimize."
That, Chaput said, has led his team to develop a strategy for identifying peptides from large combinatorial libraries created using mRNA display technology, which enables the scientists to "very easily create very large libraries" that contain as many as 1012 different peptide sequences against which targets can be screened. The large number of sequences allows them to identify peptides that can be used in synbodies without needing further optimization.
His lab is currently working on a paper to present the combinatorial library technology, Chaput said, adding that, in addition to eliminating the need for iterative rounds of optimization, this approach could also help improve the reagents' sensitivity.
"So far we've demonstrated that if you take two low micromolar peptides and put them together, you get a low nanomolar affinity reagent," he said. "With the large combinatorial libraries, the things that we've generated have sub-micromolar [sensitivity] in most cases. You put those together and you start getting picomolar-level binding, and once you're on that level of binding you're basically equivalent to the very best antibodies that are out there."
Chaput noted that he is working with his ASU colleague Josh LaBaer to fully automate the technology. The combinatorial peptide library screening could, in theory, be done in a 96-well plate format, he said, and automating the rest of the process "should be pretty straightforward."
"We've basically individually demonstrated all the … pieces for doing this on a robotic platform, but we haven't really put it all together," he said. "Now we're in the process of doing that, and we haven't really seen any major sticking points."
Given the reagents' DNA backbones, it's also possible, Chaput said, that proteins captured using them could be quantitated using next-generation sequencing, a possibility that several biotech companies that use nucleotide-based capture agents — including protein biomarker firms Pronota and Olink Bioscience — are currently exploring in hopes of taking advantage of the technology's precision and multiplexing ability (PM 5/6/2011).
"It's something that is certainly possible, and we’ve certainly thought about it," Chaput said, although he noted that it wasn't "at the top of the list" of projects for the group.
More immediately, he said, they hope to develop synbody sandwich assays, which would further improve the agents' sensitivity.
Chaput's team has submitted a patent covering the reagents, he said, 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.
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