Proteins and their interactions:

the MolBoolean™ revolution by Atlas Antibodies

Authors: Nancy Dekki PhD and Laura Pozzi PhD, Atlas Antibodies AB, Sweden

Atlas Antibodies, the Swedish Biotech company that emerged from the Human Protein Atlas project, is making waves in protein research with its MolBoolean™ technology. This innovative assay is designed to simultaneously measure free and interacting proteins within fixed cells and tissue samples, offering unparalleled insights into protein-protein interactions.

Photo: Mikael Malmqvist, PhD, Atlas Antibodies

Based in Stockholm and founded in 2006, Atlas Antibodies has grown its catalog to over 22,000 antibodies for research purposes. The development of MolBoolean™ marks a significant leap forward, combining the company's expertise in immunoassays with groundbreaking proximity detection technology.

Mikael Malmqvist, PhD, principal scientist at Atlas Antibodies, was interviewed about the MolBoolean™ technology by GenomeWeb, the New York based organization for online news.  

"MolBoolean™ is based on the In situ Proximity Ligation Assays (PLA) technology. It is a universal kit where the researchers use their own primary antibodies for their protein targets, with Atlas Antibodies supplying the secondary antibodies conjugated to unique DNA sequences. When the secondary antibodies bind to their targets, the attached DNA strands are brought into proximity and are ligated, forming a new template that can then be amplified by rolling circle amplification. Readout of the new template is done by hybridizing fluorescence- tagged oligos. In cases where the proteins are detected separately, the rolling circle amplification will produce a different oligo sequence, allowing researchers to distinguish between levels of bound and free protein." 

"The ability to measure both bound and free proteins allows researchers to normalize the number of protein-protein interactions to the total amount of 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. 

The MolBoolean™ kit includes 15 separate tubes: enzymes (nickase, ligase, polymerase), buffers, oligos for tagging and detection, secondary antibody probes and additional reagents such as blocker, and diluent.The total volume of 4.8 ml, is sufficient for approx. 120 assays in cells (40 μl/assay) and 60 assays in tissue (80 μl/assay).

From Innovation to Application: the story behind MolBoolean™

The journey of MolBoolean™ began with Professor Ola Söderberg from Uppsala University, a key inventor of the original in situ proximity ligation assay (PLA) technology almost 20 years ago. Recognizing the potential to refine PLA for broader use, Söderberg approached Atlas Antibodies with the opportunity to patent and develop MolBoolean™ into a versatile research kit. By 2019, the company secured these rights, transforming the concept into a robust and user-friendly assay.

Photo: Prof. Ola Soderberg, visiting Forskaren, the new Atlas Antibodies' headquarter

MolBoolean™, commercially launched by Atlas Antibodies in 2023, builds on PLA by allowing researchers to use their own primary antibodies, while the kit supplies secondary antibodies (mouse/rabbit) conjugated with unique DNA sequences.

When these secondary antibodies bind to their targets, their DNA strands come into proximity, forming a template for rolling circle amplification (RCA). This amplified template generates fluorescent signals that clearly differentiate between free proteins and protein complexes. Using a standard fluorescent microscope, researchers can analyze these signals to quantify both forms, enabling more precise data on protein interactions.

A Researcher’s Perspective: from Discovery to Drug Development

Professor Thomas Sakmar from Rockefeller University has embraced MolBoolean™ for his work on G-protein coupled receptors (GPCRs). His lab used the assay to validate the in vivo interactions of GPCRs with receptor activity-modifying proteins (RAMPs), findings that were pivotal for a recent study published in Science Advances (Kotliar IB, et al, (2024) Sci Adv. 2024 Aug 2;10(31):eado9959).

Sakmar highlighted the assay’s unique ability to provide both quantitative data and spatial information, something traditional methods could not achieve. For example, MolBoolean™ revealed whether GPCR complexes were located on the cell membrane or trapped in the endoplasmic reticulum, offering deeper insights into cellular processes.

Prof. Sakmar called the MolBoolean™ technology "extremely important" for validating his research and noted its potential for broader applications, including scaling it for flow cytometry-based studies.

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 Antibodies, with which his lab had a preexisting relationship, and began working with the method.

“We looked for a long time for methods that would show that the complexes actually existed in the cell membrane. The cool thing about MolBoolean™ is that it basically is 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,” Sakmar said. “that´s something you can´t do with older strategies where you just say “yes” or “no” about a complex."

Prof. 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.“

Photo: Prof. Thomas P. Sakmar, The Rockefeller University

"The MolBoolean™ technique for studying protein-protein interaction could have sweeping implications for drug development"  

Real-World Impact Technology

MolBoolean™ is already transforming how scientists approach protein interaction studies. According to Mikael Malmqvist, "its ability to measure free and bound proteins simultaneously is crucial for normalizing interaction data. This feature helps avoid errors that might arise from simple increases in protein expression levels, which can otherwise be mistaken for increased interaction rates."

In the pharmaceutical world, MolBoolean™ is generating significant interest. Companies developing drugs targeting protein degradation pathways, such as E3 ubiquitin ligases, find the technology invaluable. By measuring interactions between E3 ligases and their targets during treatment, MolBoolean™ helps researchers normalize these interactions against total protein levels, leading to clearer insights into drug efficacy. 

Rivas-Santisteban R, et al, (2024) GPR88 impairs the signaling of kappa opioid receptors in a heterologous system and in primary striatal neurons. Neuropharmacology. 2024 Nov 27:110242.doi: 10.1016/j.neuropharm.2024.110242. Epub ahead of print. PMID: 39613254.

Kotliar IB, et al, (2024) Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins. Sci Adv. 2024 Aug 2;10(31):eado9959. Epub 2024 Jul 31. PMID: 39083597; PMCID: PMC11290489.

Malmqvist M, et al, (2024) Quantifying dopamine D2 and adenosine A2A receptor interactions in rat brain using MolBoolean™ technology. #8217 Society for Neuroscience, SfN, (2024).

Raykova D, et al, (2023) A method for Boolean analysis of protein interactions at a molecular level. Nat Commun. 2023 Sep 6;14(1):5450. doi: 10.1038/s41467-023-41325-3. Erratum for: Nat Commun. 2022 Aug 13;13(1):4755. doi: 10.1038/s41467-022-32395-w. PMID: 37673885; PMCID: PMC10482831.

Rivas-Santisteban R, et al, (2023) Boolean analysis shows a high proportion of dopamine D2 receptors interacting with adenosine A2A receptors in striatal medium spiny neurons of mouse and non-human primate models of Parkinson’s disease. Neurobiol Dis. 2023 Nov;188:106341.doi: 10.1016/j.nbd.2023.106341. Epub 2023 Oct 31. PMID: 37918757.

Beyond Proximity Ligation with MolBoolean

MolBoolean™ is more than a technical innovation, it’s a tool reshaping our understanding of protein behavior and goes beyond traditional proximity ligation assays. By offering precise, normalized data and the ability to study protein interactions in situ, it is proving indispensable in drug discovery, basic research, and beyond. MolBoolean™ is poised to become a cornerstone technology in the study of protein-protein interactions.

Photo: Ingela Hofverberg, CEO at Atlas Antibodies.

 “I am very proud of MolBoolean, a cutting-edge technology developed in collaboration with leading scientists in the PLA field. This kit enables researchers to identify and measure protein-protein interactions in situ, in both fixed cells and tissue. The versatility of MolBoolean™ makes it a valuable tool for a wide range of applications, including drug discovery, biomarker identification, and understanding disease mechanisms. MolBoolean is a valuable addition to our wide range of high-quality antibodies, and we believe it will greatly benefit our customers' research” 

This blog is a summary of the article titled "Atlas Antibodies' MolBoolean Assay Offers New Insights Into Protein-Protein Interactions," published on GenomeWeb by Adam Bonislawski on October 30, 2024.