Updated: Aug 30
The study of protein-DNA interactions feeds into understanding the mechanisms of DNA replication, repair and transcription, gene expression, and the packaging of chromosomal DNA . Now, a relatively new way to study protein-DNA interactions is entering the analytical toolbox: mass photometry.
Mass photometry is ideal for this purpose because it can measure the mass of different biomolecules, such as DNA and proteins, in a single assay. Mass photometry makes it possible to study protein-DNA interactions in a native-like environment, as the measurement is performed in solution, and does not require labels, detergents or an electrical field .
Here, we present a recent study that used mass photometry to investigate protein-DNA interactions. We then speak with the paper’s first author, Ananya Acharya, who explains how she and her colleagues used mass photometry to answer unique scientific questions about DNA repair mechanisms and compares it to other techniques.
An example from the literature – investigating mechanisms of DNA damage repair
A recent study used mass photometry to study protein-DNA binding and gain insight into interactions in the machinery for homologous recombination repair of DNA damage. The study, Distinct RPA domains promote recruitment and the helicase-nuclease activities of Dna2, was published in Nature Communications .
The aim of the paper was to demonstrate how Dna2, a conserved nuclease-helicase, is recruited to the DNA double-strand break and mediates repair. Previous data had shown that Dna2 interacts with the major eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), which promotes Dna2’s enzymatic functions. However, it was unclear exactly how RPA and Dna2 interacted with one another and with DNA.
The study’s authors used mass photometry to decipher how Dna2 binds to single-stranded DNA during DNA repair and replication and whether Dna2 immediately replaces RPA on the DNA. The results revealed that Dna2 is recruited by RPA on DNA, producing an intermediate protein-DNA complex that facilitates the binding of Dna2 to DNA. This finding implies that RPA is positioned to catalyze the enzymatic activities of Dna2 that occur downstream of its recruitment (Figure 1).
Figure 1: Mass photometry showed that Dna2 only binds to DNA after the addition of RPA. Measurements were performed on four samples in triplicate. RPA alone formed a single peak at molecular weight 115 ± 27 kDa, while Dna2 formed a main peak at 166 ± 28 kDa. When both RPA and Dna2 were combined, mass peaks corresponding only to single RPA and Dna2 molecules were detected, indicating a lack of binding. However, when Dna2 and RPA were combined in the presence of DNA, a new species formed at 302 ± 67 kDa, corresponding to the combined molecular weight of the three components.
Figure source: Acharya et al., 2022 (Figure 2D), used under CC BY 4.0
Comments from the lead author
We talked to Ananya Acharya, the publication’s lead author, who is a PhD student at the Institute for Research in Biomedicine, Bellinzona and the Institute of Biochemistry, ETH Zurich. She told us how mass photometry was useful in her study, and gave her perspective on the technology’s unique features and complementarity to other techniques.
The interview responses have been edited for length and clarity.
Ananya Acharya Recombination Mechanisms PhD Student, USI Switzerland On Twitter: @AnanyaAcharya26
“Mass photometry helped us answer a longstanding question of whether DNA, protein A and B bind together or not.”
Ananya began using mass photometry one year ago, after hearing about it from her collaborator, Professor Ralf Seidel (Peter Debye Institute for Soft Matter Physics, Universität Leipzig, Germany). Seidel's group, composed of biophysicists, was already using mass photometry. Additionally, Dr. Kristina Kasaciunaite and Dr. Martin Göse from Prof. Seidel's group established the key mass photometry experiments in the study.
“At the time, we were trying to address a key problem in our research, which we were unable to solve with our current technologies. This is when Prof. Ralf Seidel told us about an upcoming, cool technology (mass photometry), explaining the physics and the principles behind it.”
Mass photometry enables the accurate mass measurement of different biomolecules in solution, using light. Ananya and her colleagues study DNA repair mechanisms and primarily use mass photometry to measure protein-DNA interactions. It enabled them to address a key question in their field.
“Mass photometry helped us answer a longstanding question of whether DNA, protein A (RPA) and B (Dna2) bind together or not. Previous research claimed that protein B replaced protein A on top of DNA. But with mass photometry we showed that protein B sits on top of DNA in the presence of protein A. We thought that since they both bind together, A is working synergistically with B, and this helped us proceed with the research towards that direction.”
Ananya compared mass photometry to other techniques she has used to study protein-DNA interactions, emphasizing that mass photometry has the advantages of being a fast, simple, label-free technique that requires minimal sample.
“Typically, we use the electrophoretic mobility shift assay (EMSA) technique to study protein-DNA interactions. In terms of experimental handling, EMSA can be very demanding: the sample needs to be kept cold, it is time-consuming, it requires a lot of material and occasionally the interactions cannot be kept stable throughout the experiment.
By contrast, mass photometry is very fast, it requires minimal sample, and it gives us instant answers from a single-molecule perspective. Additionally, we use other methods to study protein-DNA binding, which are not label-free, such as microscale thermophoresis (MST). This technique may require a lot of starting material for certain experiments. So, mass photometry is a step forward as we need to make less compromises to execute such types of experiments.”
Ananya also mentioned another advantage of mass photometry that is useful in her research: the software that comes with Refeyn’s instruments, DiscoverMP, enables her to easily explore and plot her results.
“The software is very interactive. I can do a lot of things like changing how the graph looks or merging repeated measurements and plotting an average.”
She added that she can intuitively interpret the mass distribution results without advanced data analytics knowledge.
“As a biochemist, I am not particularly experienced in dealing with large datasets. Although mass photometry is a biophysical technique, it measures the masses of proteins and DNA, which are outputs of biochemical experiments, and allows us to study their complex interplay. The fact that it is so accessible and user friendly helps me perform the experiments myself, which makes me more independent and saves time.”
Ananya is now using mass photometry to study protein-protein interactions, as it can provide information on a protein’s quaternary structure (oligomeric state) as well as binding to other proteins.
“In my current project, I use mass photometry to study protein-protein interactions. I am trying to understand if there is a specific protein that can alter the oligomeric states of a second protein and whether there are factors that can alter their interaction or oligomeric states.”
Using mass photometry, she discovered that one of the proteins she studied had different oligomeric states than expected based on previous experiments. This finding opened up a new direction for her research.
“Previous gel filtration chromatography experiments indicated that one of the proteins I studied was a hexamer, according to the marker. I subsequently did the same experiment using mass photometry and I realized that my protein exists in various oligomeric forms. We are now exploring different functions that can be attributed to these forms. This revelation would not be possible without mass photometry!”
Here you can learn about the basics of mass photometry and the main advantages it offers for the analysis of biomolecules.
This note describes in detail how to use mass photometry to measure the mass, purity and relative abundance of DNA, in the range of 100 to 5000 base pairs.
 R. A. C. Ferraz, A. L. G. Lopes, J. A. F. da Silva, D. F. V. Moreira, M. J. N. Ferreira, and S. V. de Almeida Coimbra, “DNA–protein interaction studies: a historical and comparative analysis,” Plant Methods, vol. 17, p. 82, Jul. 2021, doi: 10.1186/s13007-021-00780-z.
 R. Asor and P. Kukura, “Characterising biomolecular interactions and dynamics with mass photometry,” Current Opinion in Chemical Biology, vol. 68, p. 102132, Jun. 2022, doi: 10.1016/j.cbpa.2022.102132.
 A. Acharya et al., “Distinct RPA domains promote recruitment and the helicase-nuclease activities of Dna2,” Nature Communications, vol. 12, no. 1, Art. no. 1, Nov. 2021, doi: 10.1038/s41467-021-26863-y.