Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Researchers have devised multiple strategies for detecting and characterizing the interactions of proteins with other proteins, nucleic acids, and small molecules. They’ve got genetic approaches, such as the yeast two-hybrid assay (Y2H) of protein-protein interaction. And they’ve got biochemical alternatives, such as fluorescent labeling.
But when researchers want to study a given interaction in deeper detail, and especially if they must detail its kinetics, neither approach will do. Y2H and related methods require genetic fusions, which may alter the behavior the native molecule, and are qualitative rather than quantitative approaches. Fluorescent labeling strategies require chemical modification, which can alter molecular behavior, especially of small molecules.
Increasingly, researchers interested in a deep dive into protein biochemistry—and particularly in drug development—are turning instead to so-called label-free interaction assays. As the name implies, these methods work on native proteins, leveraging subtle changes in physical properties to record their interactions with other molecules in their environment.
Here are some of the more popular options.
Surface plasmon resonance
Most label-free methods exploit the properties of light to detect biomolecular interactions. The most widely adopted method, surface plasmon resonance (SPR), effectively detects differences in refractive index. Molecules interacting on a gold surface atop a prism change the electromagnetic properties of that surface such that the angle at which light bounces off it is also changed, causing an effect akin to sticking a straw into a glass of water. The magnitude of the effect is proportional to the extent of the interaction.
As explained here, the actual mechanics of SPR are considerably more complex than that, of course. The process involves a particular angle of incident light at which the light no longer bounces off the gold surface but rather reflects within the gold-glass interface, producing a total-internal reflection condition. This “gap” in reflection angle is what SPR instruments measure, and more specifically, its change over time.
“This change of resonant angle can be monitored noninvasively,” explains Fredrik Sundberg, global director for strategic customer relations and market development at GE Healthcare Life Sciences, which commercializes the popular Biacore™ line of SPR instruments. And it can be monitored continuously, in real-time and in any type of sample. “We are not dependent on color shift or complex matrixes.”
SPR instruments often employ fluidics to flow reagents past one another, enabling researchers to measure kinetic parameters, such as on-rates, off-rates, affinity and so on. Such details are especially valuable in drug discovery, where chemists can use the data to make informed decisions about which molecules to progress and which to hold back. One Biacore client, for instance, uses the technology to “triage their high-throughput [small molecule] screening workflow,” Sundberg says, “using our label-free technology as a filter.” Another client uses it to screen for antibody therapeutics.
“You can get information about how the drug acts with a target, how fast is the interaction, how strong is the interaction and how specific is the interaction,” Sundberg says. “You get all that information with one single technique.”
GE offers several instruments under its Biacore brand. The Biacore T200 has four flow channels for testing four interactions or conditions simultaneously. The higher-throughput Biacore 4000 can measure 16 reactions at once. Using a plate hotel, “you can look at 4,000 interactions in 24 hours,” Sundberg says.
With two sets of six flow channels arranged in a criss-cross pattern, Bio-Rad Laboratories’ ProteOn™ XPR36 can monitor 36 biomolecular interactions at once, says Ruben Luo, a Product Manager in the Protein Function Division at Bio-Rad Laboratories. That arrangement, he says, gives researchers considerable flexibility in experiment design and throughput.
“With this configuration they can do whatever kind of experiment they want: Kinetics in one shot, six-by-six screening [i.e., either six analytes tested against six targets, or a pair of interaction partners both tested for each of six conditions], or immobilizing 36 different proteins to make a protein array.”
Epic technology
Corning’s Epic® technology relies on a different property of light. Instead of reflected light angle as in SPR, Epic measures changes in reflected wavelength as molecules interact on the surface of a resonant waveguide grating, explains David Randle, applications development manager at Corning Life Sciences. “As a small molecule”—or protein—“binds to a protein target [on a sensor surface], it leads to shifts in the reflected wavelengths of light.”
As with SPR, the magnitude of the change is proportional to the extent of binding, and even very small changes can be detected, says Randle. “You can reliably measure changes as small as 1 to 2 picometers of shift in reflected wavelength, which correlates to the binding of a 50 to 100 Da molecule to a protein,” he says.
But unlike SPR, Epic measures interactions not in a flow cell but in the wells of a special microtiter plate. Thus, it provides “static read” bulk measurements rather than real-time kinetics data, albeit at considerably higher throughput than SPR, says Randle.
“We see ourselves as a technology that complements nicely the capabilities of Biacore [SPR],” he says. “We see ourselves as a workhorse for screening, with plates up to 1,536 wells.”
Corning offers Epic in both an ultra-high-throughput “freezer-sized” instrument with on-board liquid handling and a smaller benchtop unit that lacks the liquid-handling capabilities. Epic is also available on Revvity’s EnSpire® multimode microplate reader.
Bio-layer interferometry & more
Other companies are pursuing different detection modalities. ForteBio’s bio-layer interferometry (BLI) technology measures binding-induced changes in light interference patterns.
As explained on the company web site, BLI “is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer.” The company’s instruments dip those sensors into a solution of interacting protein, a process it calls “Dip and Read™.” “Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time.”
Maven Biotechnologies is developing technology based on yet another parameter, light polarization. Called “label-free internal reflection ellipsometry” (LFIRE™), the technique effectively enables label-free imaging of surface thickness in a microtiter well, explains David Ralin, the company’s chief technology officer. “You can see an image of the antibody or antigen array directly… and as the spots capture material, you see the thickness change in the spots, pixel by pixel in real time.”
Not all companies are pursuing light-based strategies. Attana has commercialized systems based on quartz crystal microbalances (QCM). According to the company, the QCM acts essentially like a tiny scale, measuring changes as reagents flow past. “An applied AC-potential causes the quartz crystal to vibrate at its resonance frequency. As molecules flown over the crystal bind to the surface, the vibration frequency changes.” ACEA Biosciences’ xCELLigence system exploits electrical impedence changes caused by cellular responses to protein-protein and protein-drug interactions, among other things.
Clearly, those interested in label-free analyses have a lot of options. But don’t plan on retiring your labeling protocols just yet, says Randle. Though they don’t analyze proteins in their native format, label-based methods are well established and validated by the community, he points out. They also are much higher throughput, and many researchers still use labeled approaches to drive early screens, following up with orthogonal label-free methods to gather more rigorous characterization data on promising hits.
“You have to look at label-free as complementing label-based techniques,” he says.
Image: Detail of a surface plasmon resonance configuration. [Credit: Sari Sabban, University of Sheffield; Source]