Of the multitudinous ways of detecting protein-protein and protein-drug interactions, few if any satisfy all researchers’ desired criteria: that the method should be specific, require minimal protein engineering to construct, and be applicable to many types of targets and amenable to high-throughput experiments, to name just a few requirements.
A team of researchers at the University of Bonn and the University of Texas may have come closer to fulfilling at least two of those demands. Reporting in the July issue of Nature Biotechnology, Michael Famulok of the University of Bonn and his colleagues describe their technique for detecting protein-protein and protein-drug interactions using a ribozyme switch, in conjunction with an aptamer capture agent, to create an assay that is potentially applicable to many types of protein targets, and also potentially amenable to high-throughput experiments.
Cleaving the Ribozyme
Famulok’s team designed the technique to take advantage of a property of two particular types of ribozymes, the so-called hairpin and hammerhead ribozymes. In general, a ribozyme is a RNA molecule capable of catalyzing simple nucleic acid reactions, but the hairpin and hammerhead ribozyme are self-cleaving, that is, when triggered, two arms of the ribozyme complex either associate closely or separate from each other.
Taking advantage of this, the researchers developed a scheme for fusing an aptamer — a single-stranded nucleic acid capable of folding into intricate shapes — with the ribozyme, in such a way as to allow the aptamer to control whether or not the ribozyme cleaves itself. In one version of the technique, the aptamer acts to prevent the ribozyme from cleaving, except when a protein target specifically binds to the aptamer. When that happens, the ribozyme is free to cleave.
But how to make this protein-aptamer interaction detectable? Famulok’s team chose to attach a fluorescent reporter to one arm of the ribozyme complex, and a quencher to the other arm. When the aptamer has no protein to interact with, the two arms are physically close, quenching the fluorescence. Conversely, when a protein comes along to interact with the aptamer, the ribozyme complex cleaves, allowing one of the arms to fluoresce.
This method for creating what Famulok calls “reporter ribozymes” can be made to perform more intricate experiments as well. In one example described in the paper, Famulok shows that a protein target, once bound to the aptamer of the reporter ribozyme, can be displaced by a small molecule compound that interacts specifically with the protein. When this occurs, the fluorescence of the reporter ribozyme is again quenched, giving a readout of the protein-drug interaction. Likewise, Famulok suggests that this approach is also applicable to querying protein-protein interactions.
Famulok claims his method has several advantages over comparable techniques. For one, the technique is compatible with a 96-well plate format. In addition, it is easily transferable to many types of protein targets, given that the technology exists for creating libraries of up to 1015 structurally distinct aptamers. Using this technology, known as SELEX, an aptamer could be found for any protein or drug target.
“Typically, screening targets is tedious because each assay must be tailor made for each target,” he said. “This technique is modular, like LEGO, because you can quickly obtain a specific binder against any protein target.”
However, the technique does have limitations, as Famulok admits. The ability of the reporter ribozyme to detect an interaction is dependent on a given concentration of the target protein in the assay, with a lower limit of “higher nanomolar concentrations,” Famulok said.
Larry Gold Weighs in
In addition, as SomaLogic CEO Larry Gold points out in an accompanying commentary in Nature Biotechnology, the aptamer might not necessarily bind to the site on the protein that is active in vivo. “Aptamers (or antibodies) show epitope dominance, and there is no reason to assume that the dominant epitopes for these [capture agents] will overlap the active sites of the proteins,” he wrote.
Nevertheless, in future experiments Famulok hopes to demonstrate the general applicability of the reporter ribozyme technique, by increasing the number of different aptamers to perform multiplexed assays.
Famulok also plans to show that his team can use other types of capture agents, such as RNA molecules, to develop assays for therapeutically important RNA-protein interactions.