NEW YORK – Swedish life science firm Atlas Antibodies has developed a new kit for in situ measurements of protein-protein interactions, offering advancements on proximity ligation assay (PLA) technology.
Called MolBoolean, the product allows researchers to distinguish between target proteins present in a sample bound together in a complex and those same proteins present as free, unbound molecules.
According to the company, the method is seeing uptake in areas like drug discovery and development and for research into conditions including Parkinson's disease.
Mikael Malmqvist, principal scientist at Atlas Antibodies, said that the MolBoolean assay emerged from a strategy the company embarked on in recent years to develop and offer immunoassay products leveraging its antibody catalog. Stockholm-based Atlas was founded in 2006 to commercialize antibodies used in the Human Protein Atlas project. The company currently offers more than 22,000 antibodies for research applications.
Several years ago, Atlas was approached by Ola Söderberg, the inventor of the MolBoolean technology and a professor in the department of pharmaceutical biosciences at Uppsala University. "Professor Söderberg asked if we wanted to reserve the rights to patent the technology and develop it into a kit product, and we thought it was a great add-on to our existing catalog," Malmqvist said. Atlas secured the rights to the technology in 2019.
MolBoolean is based on the PLA technology developed almost 20 years ago by Uppsala scientists including Söderberg. PLA uses pairs of antibodies attached to unique DNA sequences to detect proteins of interest. When the antibodies bind to their targets, the attached DNA strands are brought into proximity and ligated, forming a new template that can then be amplified by rolling circle amplification. Readout of the new template can be done via PCR or next-generation sequencing or, in the case of in situ assays like MolBoolean, by hybridizing fluorescence-tagged oligos.
In situ PLA assays have long been used to observe protein-protein interactions in cells and tissue samples, with Swedish life science firm Navinci offering a commercial version of the technology for spatial proteomics research. MolBoolean advances on traditional in situ PLA in that it allows researchers to simultaneously measure the amount of two target proteins bound together in a complex and the amount of these target proteins existing as free, unbound protein in the sample.
In the MolBoolean assay, when two target proteins are bound together in a complex, the antibodies targeting them will be brought into close proximity, enabling the production of a specific oligo sequence via rolling circle amplification. In cases where the proteins are detected separately, the rolling circle amplification will produce a different oligo product, allowing researchers to distinguish between levels of bound and free protein.
Researchers use their own primary antibodies to their protein targets, with Atlas supplying the secondary antibodies conjugated to oligos that bind those primary antibodies to produce the MolBoolean signal. Malmqvist said that to effectively distinguish between free and bound proteins, users need a fluorescence microscopy system capable of spatial resolution of at least 100 nanometers.
The ability to measure both bound and free protein allows researchers to normalize the number of protein-protein interactions to the total amounts of the target proteins in a sample. This can help prevent misinterpretation of protein interaction data in situations where, for instance, an increase in interaction signal stems from higher expression of the target proteins as opposed to increased interaction rates, Malmqvist said.
He said that Atlas has received substantial interest from pharma companies in the targeted protein degradation space, in which small molecule agents are used to connect E3 ubiquitin ligases involved in the degradation and elimination of nonnative misfolded proteins to protein targets involved in disease. In this case, MolBoolean can be used to measure levels of interaction between E3 ligases and protein targets following drug treatment while normalizing against the amount of free E3 ligase and target protein present.
Thomas Sakmar, a professor at the Rockefeller University, has been using MolBoolean for his research into G protein coupled receptors (GPCRs). In a recent study in Science Advances, he and his colleagues used the approach to confirm in vivo interaction of GPCRs and receptor activity-modify proteins (RAMPs) — which can impact the function of GPCRs — that were found to bind in a large-scale in vitro screening assay.
Sakmar noted that while the in vitro screen provided evidence that the observed GPCR-RAMP pairs could bind, its didn't conclusively demonstrate that these complexes actually formed in cells.
"We looked for a long time for methods that would show that the complexes actually existed in the cell membrane," he said. Upon coming across the paper describing the MolBoolean technique, which Söderberg and his colleagues published in Nature Communications in 2022, Sakmar reached out to Atlas, with which his lab had a preexisting relationship, and began working with the method.
The MolBoolean work was "extremely important" to the Science Advances work as it allowed the researchers to validate the in vitro screening approach, Sakmar said.
"The cool thing about MolBoolean is that it is basically a colocalization assay where you have very good quantitative readouts for each component, and you can use that information to calculate an apparent affinity constant for complexes," he said. "That's something you can't do with [older] strategies where you just say 'yes' or 'no' about a complex."
Sakmar said the spatial information provided by the technique also offers insights, citing his work on GPCR-RAMP interactions.
"Say the [GPCR] needs a RAMP to get to the cell surface, but if the RAMP is not there or is defective, then the [GPCR] is still going to get synthesized, but it will get stuck in the endoplasmic reticulum, for example," he said. "So, knowing the localization of the [GPCR] is interesting."
He suggested the approach could be useful across a range of applications including, for instance, observing changes in protein interactions in response to cell stress or other perturbations.
Sakmar said his lab is also interested in devising ways to scale up the MolBoolean approach, perhaps by converting it to a flow cytometry-based assay.