Revolutionizing Protein Research: Insights Into MolBoolean™ With Professor Ola Söderberg

I have been working with method development for many years. I joined Ulf Landegren’s group almost 20 years ago, and since then, we have been developing many different methods, like in situ proximity ligation assay (PLA), which was first described in the paper “Direct observation of individual endogenous protein complexes in situ by proximity ligation”, published in Nature Methods in 2006. For the last 15 years, I have headed a research group focusing on method development and applying these methods in tumor biology.

Method development is crucial to making new leaps in science. Novel methods provide researchers with new research tools, opening new avenues of what can be explored. Yet, the creative side of method development is rarely described in literature, how methods are conceived. In the 2024 article, “The method developer’s guide to oligonucleotide design”, published in Expert Review of Proteomics, I describe my approach to method development.

I worked on developing MolBoolean for eight years, in order to be able to determine protein levels in individual cells while also identifying the proportion of these proteins that bind to each other, and I described the method in “A method for Boolean analysis of protein interactions at a molecular level”, published in Nature Communications in 2022. 

If you compare MolBoolean to methods like in situ PLA, which measures the amount of protein complexes, MolBoolean also gives information about how much free protein you have in a cell, giving a better understanding of the cells’ activity status and communication. It can be compared to evaluating a restaurant — ten positive reviews provide some information, but it’s much more valuable to know whether the question was answered by ten people or a thousand.

The MolBoolean assay is a two-day workflow consisting of seven steps, designed to determine whether target proteins are interacting (denoted as AB) or present individually (A or B). On day one, the assay begins with a blocking step, which minimizes non-specific binding in the sample. Once this is complete, primary antibodies are added and allowed to incubate, attaching themselves to the specific target proteins of interest.

The second day involves a series of steps to label, amplify and detect the proteins. First, proximity probes are added, which bind to the primary antibodies that are already attached to the target proteins. These probes will then bind DNA circles, creating a foundation for the subsequent steps.

Next, a nicking enzyme is introduced, cutting the DNA circle to prepare it for tagging. Following this, specific DNA oligonucleotides (tags) are added to mark the proteins uniquely. These DNA segments are then ligated (joined together), recreating DNA circles with one or two tags incorporated (representing free proteins or protein complexes).

In the final stages, rolling circle amplification (RCA) is performed to amplify the signal from the DNA, making it easier to detect. The process concludes with the detection step, which reveals whether the target proteins are interacting (AB) or exist separately as A and B.

MolBoolean is compatible with protocols already established in the lab for primary antibodies. This flexibility allows researchers to seamlessly integrate MolBoolean into their existing workflows without requiring the purchase of specialized antibodies, making it a convenient and adaptable tool for studying protein interactions and free protein levels. The specificity of the primary antibodies can affect the accuracy of MolBoolean results. It is important to validate the primary antibodies to ensure that they specifically target the proteins of interest before applying the MolBoolean technology.

Compared to methods like in situ PLA, which measures the amount of protein complexes, MolBoolean also gives you information about how much free protein you have in a cell. There are other methods, like FRET, where you can measure both free and complex bound protein, but MolBoolean provides signal amplification by RCA, so you can detect single molecules.

MolBoolean has the advantage that you can look in single cells. You can see the amount of protein and protein complex in the cell. So that is a tool that you can use to study signal transduction. For example, if you have free protein and then you have the signal from outside, it's a relay of protein-protein interactions that goes down to the nucleus, for example, where you induce the expression of target genes. So, it's better to understand basic functions in the cell.