A5001187388- DNA and Nucleic Acid Chemistry
- Advanced biosensing and bioanalysis techniques
- RNA and protein synthesis mechanisms
- RNA Interference and Gene Delivery
- Genomics and Chromatin Dynamics
- Advanced Fluorescence Microscopy Techniques
- Bacteriophages and microbial interactions
- Single-cell and spatial transcriptomics
- Bacterial Genetics and Biotechnology
- Advanced Electron Microscopy Techniques and Applications
- Advanced Biosensing Techniques and Applications
- HIV Research and Treatment
- Molecular Biology Techniques and Applications
- Tuberculosis Research and Epidemiology
- Plant Molecular Biology Research
- Lipid Membrane Structure and Behavior
- Nanopore and Nanochannel Transport Studies
- Mycobacterium research and diagnosis
- Viral Infections and Vectors
- Genetics, Bioinformatics, and Biomedical Research
- Cell Image Analysis Techniques
- DNA Repair Mechanisms
- CRISPR and Genetic Engineering
- Clostridium difficile and Clostridium perfringens research
- Plasmonic and Surface Plasmon Research
Indian Institute of Science Bangalore
2021-2025
Max Planck Institute of Biochemistry
2018-2024
Ludwig-Maximilians-Universität München
2018-2021
Center for NanoScience
2018-2021
Delft University of Technology
2016-2019
It has been hypothesized that SMC protein complexes such as condensin and cohesin spatially organize chromosomes by extruding DNA into large loops. We directly visualized the formation processive extension of loops yeast in real time. Our findings constitute unambiguous evidence for loop extrusion. observed a single complex is able to extrude tens kilobase pairs at force-dependent speed up 1500 base per second, using energy adenosine triphosphate hydrolysis. Condensin-induced extrusion was...
To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access location and interaction all biomolecules. Significant advances were achieved super-resolution microscopy, but such methods are still far from reaching multiplexing capacity proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that capable achieving virtually at better than 15 nm resolution. Using...
The nuclear pore complex (NPC) is the gatekeeper for transport in eukaryotic cells. A key component of NPC central shaft lined with intrinsically disordered proteins (IDPs) known as FG-Nups, which control selective molecular traffic. Here, we present an approach to realize artificial mimics that allows controlling type and copy number FG-Nups. We constructed 34 nm-wide 3D DNA origami rings attached different numbers NSP1, a model yeast FG-Nup, or NSP1-S, hydrophilic mutant. Using (cryo)...
Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds molecular species are still elusive. Here, we introduce an entirely new approach multiplexed microscopy by designing blinking behavior targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding silico vitro using DNA origami structures, show applicability RNA protein...
Abstract The DNA double helix structure is stabilized by base-pairing and base-stacking interactions. However, a comprehensive understanding of dinucleotide energetics lacking. Here we combined multiplexed DNA-based point accumulation in nanoscale topography (DNA-PAINT) imaging with designer nanostructures measured the free energy base stacking at single-molecule level. Multiplexed enabled us to extract binding kinetics an imager strand without additional DNA-PAINT data showed that single...
The three-dimensional organization of DNA is increasingly understood to play a decisive role in vital cellular processes. Many studies focus on the DNA-packaging proteins, crowding, and confinement arranging chromatin, but structural information might also be directly encoded bare itself. Here, we visualize plectonemes (extended intertwined structures formed upon supercoiling) individual molecules. Remarkably, our experiments show that sequence encodes structure supercoiled by pinning at...
Super-resolution microscopy is transforming research in the life sciences by enabling visualization of structures and interactions on nanoscale. DNA-PAINT a relatively easy-to-implement single-molecule-based technique, which uses programmable transient interaction dye-labeled oligonucleotides with their complements for super-resolution imaging. However, similar to many imaging approaches, it still hampered subpar performance labeling probes terms large size limited efficiency. To overcome...
DNA supercoiling crucially affects cellular processes such as replication, gene expression, and chromatin organization. However, mechanistic understanding of the related DNA-processing enzymes has remained limited, mainly due to lack convenient experimental tools probe these phenomena. Here, we report a novel high-throughput single-molecule assay for real-time visualization supercoiled molecules, named ISD (Intercalation-induced Supercoiling DNA). We use an intercalating dye induce...
Condensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so largely unknown. We show condensin's has dual role in chromosome condensation. Mutation of one site impairs condensation, while mutating the second results hyperactive condensin compacts DNA faster than wild-type, both vivo and vitro. Whereas drives loop formation, involved formation more stable higher-order Z structures. Using I, we reveal II not intrinsically needed for shortening...
Abstract The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is nucleoid-associated that interacts with DNA to create biomolecular condensates live bacteria. Purified can also rapidly form large complexes when combined vitro. However, the mechanism allows these nucleate on remains unclear. Here, we examine how topology influences formation Dps–DNA complexes. We find supercoils offer most preferred...
SUMMARY To fully understand biological processes and functions, it is necessary to reveal the molecular heterogeneity of cells even subcellular assemblies by gaining access location interaction all biomolecules. The study protein arrangements has seen significant advancements through super-resolution microscopy, but such methods are still far from reaching multiplexing capacity spatial proteomics. Here, we introduce Secondary label-based Unlimited Multiplexed DNA-PAINT (SUM-PAINT), a...
The xenogeneic silencer protein Lsr2 from Mycobacterium tuberculosis plays a critical role in its survival and pathogenesis. is nucleoid-associated that interacts with DNA vivo regulates many genes. Purified forms nucleoprotein filaments molecules leading to highly compacted conformations. However, the physical mechanism underlying Lsr2-mediated compaction, resulting gene regulation, remains elusive. We employed combination of biochemical assay, single-molecule imaging, molecular dynamics...
<title>Abstract</title> The infectious microbe <italic>Staphylococcus aureus</italic> releases an array of cytotoxic pore-forming toxins (PFTs) that severely damage the cell membrane during bacterial infection. However, interaction interfaces between host and toxin were hardly explored. So far, there are no pore, intermediate structures these available in presence bio-membrane, which could elucidate mechanism. Therefore, we investigated structure different conformational states this...
Finding the target site and associating in a specific orientation are essential tasks for DNA-binding proteins. In order to make search process as efficient possible, proteins should not only rapidly diffuse but also dynamically explore multiple local configurations before diffusing away. Protein flipping is an example of this second that has been observed previously, underlying mechanism remains unclear. Here, we probed protein at single molecule level, using HIV-1 reverse transcriptase...
Abstract Current optical super‐resolution implementations are capable of resolving features spaced just a few nanometers apart. However, translating this spatial resolution to cellular targets is limited by the large size traditionally employed primary and secondary antibody reagents. Recent advancements in small efficient protein binders for microscopy, such as nanobodies or aptamers, provide an exciting avenue future; however, their widespread availability still limited. To address issue,...
Improving labeling probes for state-of-the-art super-resolution microscopy is becoming of major importance. However, there currently a lack tools to quantitatively evaluate probe performance regarding efficiency, precision, and achievable resolution in an unbiased yet modular fashion. Herein, we introduce designer DNA origami structures combined with DNA-PAINT overcome this issue quantification using antibodies nanobodies as exemplary probes. Whereas current assessment binders mostly...
Abstract The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is nucleoid-associated that interacts with DNA to create biomolecular condensates live bacteria. Purified can also rapidly form large complexes when combined vitro . However, the mechanism allows these nucleate on remains unclear. Here, we examine how topology influences formation Dps-DNA complexes. We find supercoils offer most preferred...
RHIM is a protein motif facilitating the assembly of large signaling complexes triggering regulated cell death. A few DNA viruses employ viral RHIMs mimicking host and counteract death by interacting with RHIM-proteins to alleviate antiviral defenses. Whether RNA operate such remains unknown. Here, we identified in Nsp13 SARS-CoV-2 other bat viruses, providing basis for bats as hosts their evolution. promoted RNA-binding channel-dependent However, more critical human than bat-derived Tb1 Lu...