Periodic Reporting for period 4 - nanoDNArepair (Next Generation Nanofluidic Devices for Single Molecule Analysis of DNA Repair Dynamics)
Reporting period: 2024-10-01 to 2025-03-31
Double-strand breaks (DSBs) in chromosomes are one of the many types of DNA damage that can occur via normal cellular processes or environmental exposures. DSBs can lead to cell death if left unrepaired; if not repaired properly, they can cause deletions, translocations and fusions in the DNA. By its nature, a DSB is located at the end of a DNA strand, rendering it difficult to study with conventional single DNA molecule methods that involve anchoring the DNA at its ends to hold it in place. The EU-funded nanoDNArepair project is creating a nanofluidics platform to study DNA repair at a single-molecule level. After trapping the DNA within a nanofluidic channel, proteins can be added or removed through a slit perpendicular to the length of the channel. The pioneering setup will enable access to unprecedented spatio-temporal details of DNA repair processes on genomic length DNA on single DNA molecules.
When concluding the project we have performed several studies on repair of broken DNA in both eukaryotes and bacteria. We have also expanded the technology to other DNA-protein systems and amyloid fibrils.
When concluding the project we have performed several studies on repair of broken DNA in both eukaryotes and bacteria. We have also expanded the technology to other DNA-protein systems and amyloid fibrils.
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)
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)
Our work is beyond state of the art from several aspects, both methodological and biological.
We are working on further developing the nanofluidics assay to allow complex dynamic reactions to be investigated. We are taking steps in this direction. We have observed reactions promoted by proteins where two DNA ends are bridged, which is the first step in DNA repair. We are working on showing this for repair proteins and we are expanding the capabilities of the dynamic nanofluidic device to study complex reactions. During the last part of the project we have designed and fabricated a new nanofluidic device that will allow addition of several different reagents. This is the next step in device design. The other important step forward is the implementation of a multiplexed nanofluidic device that is automated and multiplexed, which means that data can be collected in a much faster way.
We have also revealed new biological insights into repair of broken DNA. For bacteria the NHEJ reaction had not been investigated on the single DNA molecule level before. We revealed important details about the mechanics of DNA tethering and also proposed biological mechanisms that must be at play in order for NHEJ to be efficient. For CtIP in human cells we explored the interaction of CtIP with DNA on its own where we demonstrated that it can bridge DNA to force two DNA ends to come in close proximity, a crucial step in repair of broken DNA. The biological role of this is yet to be confirmed. During the last stages of the project we have worked with other protein complexes focusing on DNA repair , in particular the MRN complex, and bacterial genome organization, focusing on the bacterial protein Rok. For MRN we identified a potential role for the NBS subunit in how the repair will proceed. For Rok we discovered a new function that might be relevant for DNA repair in bacteria.
We are working on further developing the nanofluidics assay to allow complex dynamic reactions to be investigated. We are taking steps in this direction. We have observed reactions promoted by proteins where two DNA ends are bridged, which is the first step in DNA repair. We are working on showing this for repair proteins and we are expanding the capabilities of the dynamic nanofluidic device to study complex reactions. During the last part of the project we have designed and fabricated a new nanofluidic device that will allow addition of several different reagents. This is the next step in device design. The other important step forward is the implementation of a multiplexed nanofluidic device that is automated and multiplexed, which means that data can be collected in a much faster way.
We have also revealed new biological insights into repair of broken DNA. For bacteria the NHEJ reaction had not been investigated on the single DNA molecule level before. We revealed important details about the mechanics of DNA tethering and also proposed biological mechanisms that must be at play in order for NHEJ to be efficient. For CtIP in human cells we explored the interaction of CtIP with DNA on its own where we demonstrated that it can bridge DNA to force two DNA ends to come in close proximity, a crucial step in repair of broken DNA. The biological role of this is yet to be confirmed. During the last stages of the project we have worked with other protein complexes focusing on DNA repair , in particular the MRN complex, and bacterial genome organization, focusing on the bacterial protein Rok. For MRN we identified a potential role for the NBS subunit in how the repair will proceed. For Rok we discovered a new function that might be relevant for DNA repair in bacteria.