Please note: This article has been updated to correct an error in the previous version. The research conducted by the scientists at the Korea Advanced Institute of Science and Technology was published in the July 1 issue of Science, not Nature as previously stated.
South Korean researchers have developed a technology that combines fluorescent imaging with magnetic manipulation of labeled magnetic nanoprobes, and have shown how it can be used in a genome-wide expression screen for small molecule target identification.
Target ID and validation are becoming big application areas for high-content screening and automated imaging among drug makers. The technique, nicknamed MAGIC (magnetism-based interaction capture), may mesh well with HCS and automated imaging because it is also image-based — it would merely add magnetic manipulation of cellular entities to the fold.
In the short term, the researchers, from the Korea Advanced Institute of Science and Technology and startup biotech CGK, said they hope to commercialize the technology as a tool for high-throughput screening to identify multiple protein targets of a drug, protein modification, and protein-protein interactions in living cells.
"Our current efforts are [being] put into the final optimiz[ation] and tuning of every single step in MAGIC for high-throughput screen[ing] of molecular interactions."
In the longer term, the technique may also be useful for imaging interactions of molecules in cells in the living body when combined with a form of MRI, Tae Kook Kim, lead author on the paper and a researcher at the Korea Advanced Institute, wrote in an e-mail to CBA News last week.
The researchers published the results of their research in the July 1 issue of Science in an article entitled "A Magnetic Nanoprobe Technology for Detecting Molecular Interactions in Cells."
The MAGIC technology is based on iron superparamagnetic nanoparticles, each "almost the size of native large proteins," Kim said. Nanoparticles such as these have been used for several years now in biomedical research, most notably as a method to track cells inside of small animals using magnetic resonance imaging.
Kim and colleagues, however, eschewed the MR imaging capabilities of the probes, instead focusing on their magnetic properties as a way to move proteins around inside of a cell.
As detailed in the Science article, to perform the general MAGIC technique, the researchers conjugated streptavidin to nanoparticles in order to attach biotinylated small molecules to their surfaces. They then used transducable fusogenic peptides to achieve cellular uptake of the particles. Once the particles were inside the cells, intracellular proteins would bind to the small molecules on the particles' surface.
"Thus, when a magnetic field is applied, the [magnetic nanoprobe] and associated target protein(s) can be concentrated," the researchers wrote in the paper. "Fusion of a fluorescent probe to the target protein renders this translocation easily detectable by confocal microscopy."
The researchers conducted a number of proof-of-principle experiments in which they tracked FITC-labeled nanoparticles in cells. In one set of experiments, as a control, they transduced FITC-labeled magnetic nanoparticles in one set of HeLa cells, and luminescent semiconductor quantum dots in another. Brief application of a magnetic field to the cells resulted in noticeable translocation of the nanoparticles, but not the quantum dots.
To conduct target identification experiments using the probes, the researchers labeled probes with a specific peptide fragment known to bind caspase-3, then introduced them to cells that had been genetically engineered to express either enhanced GFP or a caspase-3-RFP construct. Application of the magnetic field resulted in translocation of the "red" signal — meaning the caspase-3 fusion constructs had bound to the peptides on the probes — but no translocation of the "green" signal.
Lastly, Kim and colleagues made retroviral eGFP-cDNA fusion protein expression libraries to use MAGIC in the systematic target identification for a bioactive small molecule. Using this method, the researchers successfully identified multiple receptors for an immunosuppressant, FK506.
Kim said he has filed for a patent on the technique in South Korea, and the researchers have now hired a law firm to take the same step in the US. Kim said that the patent is expected to be filed within the month.
Furthermore, Kim said, the technology was developed in collaboration with South Korean biotech CGK, and researchers from the two groups have discovered "novel compounds and target proteins" using MAGIC. CGK and the Korea Advanced Institute of Science and Technology are based in Daejeon.
"The next step is to make MAGIC and related progresses systematic and commercialized," Kim wrote in an e-mail. "In fact, we do have limited funds for this. Due to potential huge impacts in various areas, several companies have already contacted us. We have started to actively search for partners and funding; thus, any kind of opportunities including licensing and collaboration are widely open now." He did not detail the types of companies that have contacted him about the technology.
Kim envisions a variety of applications for MAGIC, but lists drug discovery first and foremost.
"Our current efforts are [being] put into the final optimiz[ation] and tuning of every single step in MAGIC for high-throughput screen[ing] of molecular interactions," Kim wrote. "In the near future, we will start to systematically screen target proteins binding to bioactive small molecules or small molecules binding to specific target proteins inside living human cells. Indeed, we have already made such successful several cases in aging and oncogenic processes."
Currently in drug discovery, target ID and validation are becoming big application areas for high-content screening and automated imaging. The MAGIC method may be able to mesh well with such techniques since it is image-based — it would merely add magnetic manipulation of cellular entities to the fold. Kim and colleagues used standard confocal microscopy in its experiments, but it's easy to see how some of the confocal or confocal-like HCS platforms on the market could be used to increase throughput.
Although the researchers used the transducible fusogenic peptide TAT-HA2 to induce uptake of the probes into the cells, Kim told CBA News that more efficient delivery of the particles will likely be one of the bigger challenges moving forward.
"Rapid advances in technologies for delivering a wide variety of biologically active cargos into cells both in vitro and in vivo will help to fully realize the promise of our … technology," he wrote in the e-mail.
Furthermore, although Kim told CBA News that the technique should be suitable for longer term kinetic assays in living cells, and that the particles are "biologically compatible and safe," the researchers have yet to examine the full toxicity effects on cells of prolonged incubation with the magnetic probes.
— Ben Butkus ([email protected])