We use nanofluidic tools to study DNA-protein interactions,including proteins involved in DNA repair in bacteria and eukaryotes.
The first eukaryotic protein studied was CtIP, a protein most known as a co-factor to the MRN complex but that had been suggested to also have a structural role in repairing DSBs. We demonstrated a mechanistic study how this works and how it depends on the structure of the protein. Öz et al, PNAS 2021.
We then turned to the whole MRN complex. This turned out to be more challenging but in the end we could conclude that the Nbs1 component of the MRN complex is important for the end-bridging activity. Möller et al, BBRC 2023.
For bacterial proteins we were interested in Non-Homologous End-Joining . This process consists of only two proteins, Ku and LigD, where Ku binds to DNA ends and LigD repairs the break by binding to Ku.
We presented the first single molecule study on bacterial NHEJ where we demonstrated that Ku from b. subtili can bridge two DNA ends but that LigD stabilizes this complex. We also showed that Ku threads onto DNA ends and that many Ku proteins can thread onto the same end. Öz, NAR, 2021.
Our study on bacterial NHEJ has been followed up by two studies. In the first we used Alphafold to predict the interaction interface of Ku and LigD and expressed six LigD mutants predicted to bind weaker to Ku. The results show that both domains in LigD are involved in binding to Ku and that interfering with this interaction affects the LigD function. Pavlova, et al, submitted.
We studied NHEJ in M tuberculosis where interfering with NHEJ might be important when designing new treatments for tuberculosis. We we designed and investigated mutants that interfere with the Ku-LigD interaction. Morati et al, in preparation.
For M tuberculosis we are also involved in a larger study on the structure of Ku bound to DNA. The study is mainly based on cryo-EM where Ku-filaments on DNA were studied in detail. We contributed with proteins and biochemical analysis. Zahid, et al, in revision.
We studied other DNA-binding proteins including genome organization proteins in bacteria. In our first study, we investigated the protein Rok from B subtilis. For this study we implemented a device that has ten parallel sets of nanochannels and can be handled in an automated way. We confirmed bulk experiments on Rok and discovered a novel function in annealing of DNA ends. This might be important for homologous recombination in bacteria. Pavlova et al, QRB Discovery 2025.
We studied the NC protein from the HIV-1 virus. The virus is RNA-based but it has been demonstrated that the reverse transcription starts inside the capsid, and it is therefore important that NC compacts the produced DNA. We demonstrated that NC locally compacts DNA in a phase-separation-like manner. Jiang et al, NAR 2021.
We collaborated with L. Baranello on phase separation of DNA-protein condensates. We studied Top2A and the oncogenic driver MYC. We used fluorescence microscopy to study single protein aggregates. We demonstrated how MYC affects the phase separation of TOP2A and DNA. Cameron et al, in revision.
We studied alpha-synuclein, a protein involved in Parkinson’s disease where it aggregates to form amyloid fibrils. We used single molecule analysis to investigate the interaction between amyloids and DNA and demonstrated that alpha-synuclein amyloids damage DNA by cleaving the backbone. This could be a mechanism by which alpha-synuclein causes disease. Horvath et al, ACS Chemical Neuroscience, 2025.
We used the nanofluidics to study the amyloid fibrils . Amyloid fibrils are much stiffer than DNA so we had to redesign the nanofluidic device so that it is useful for the properties of amyloid fibrils. Amyloids are difficult to analyze with single molecule techniques since most techniques require that they are anchored to a surface. Confinement in nanochannels circumvent this problem. We demonstrated that nanochannels can be used to determine the persistence length of amyloid fibrils. We could show that the persistence length is different for amyloids formed by different proteins. Sasanian et al, Nanoscale 2023.
We used the nanofluidic device to study interactions between small molecules and DNA. Focus has been on metallodrugs with potential cancer relevance designed in the group of A. Kellet in Dublin. We have one published study (O’Caroll et al, NAR 2025) and one manuscript in preparation (Poole et al)
