Biology subjects

Koenen, T.

Publications and source records attributed to Koenen, T..

7 recordsLinked to original sources

bioRxiv2026

OptiFoot: a method for recording protein footprints on DNA for microscopy and sequence analysis

A comprehensive understanding of protein-nucleic acid interactions in the crowded nuclear milieu is essential for elucidating genome function. State-of-the art methods provide the finest details of genome organization, but lack integration of imaging and sequencing modalities. We introduce OptiFoot, a fluorescence-based platform for recording protein-nucleic acid interactions in cells. OptiFoot employs an engineered sequence-unspecific N6-adenine methyltransferase that attaches diverse functional groups to DNA and RNA. When targeted to protein of interest by genetic fusion or antibody, it generates covalent high contrast fluorescent footprints that can be visualized by super-resolution microscopy and analyzed by optical mapping of single native DNA molecules, allowing complementary spatial and genomic analyses. To illustrate OptiFoot versatility, we imaged lamina-associated domains, DNA replication sites, CTCF binding sites and histone modifications in human cells and produced corresponding genome profiles. We use OptiFoot to demonstrate that majority of nucleoporin NUP153-DNA interactions occur in nucleoplasm, outside nuclear pore.

biochemistry↗

bioRxiv2025

Regioisomer-controlled red-shifted DNA probes for imaging of living tissues

Fluorescence imaging of chromatin DNA with high resolution and specificity is key to understanding cellular processes and enabling molecular diagnostics. However, choosing the best DNA probe for in vivo imaging is often a challenging task, as systematic studies investigating the biocompatibility of these molecules are lacking. Red-shifted fluorescent probes are particularly advantageous for imaging thick tissues, as light scattering decreases at red and far-red wavelengths. We synthesized and characterized the 4', 5', and 6' carboxyrhodamine regioisomers of SiR-Hoechst and 610CP-Hoechst DNA probes and assessed their performance in mice following intravenous injection. All variants entered tissues and produced stable staining that persisted for several hours ex vivo. In particular, 5' regioisomers show the highest performance, yielding bright and specific nuclear labeling. Renal clearance of the probes was nearly complete within 24 h, as indicated by fluorescence analysis of urine samples. Owing to their low cytotoxicity, high specificity, and favourable photophysical properties, these probes enabled high-quality confocal, two-photon, and STED microscopy in liver, kidney, lung, and heart tissue. Our findings extend the application of rhodamine-based DNA probes to in vivo imaging highlight their potential for deep-tissue imaging in live animals.

biochemistry↗

bioRxiv2025

A Highly Selective, Cell-Permeable Fluorescent Probe for Imaging Histone Deacetylase 6 in Live Cells

Histone Deacetylase 6 (HDAC6) plays a crucial role in diverse cellular processes, including cytoskeletal regulation, protein quality control, and stress responses; and its dysregulation is linked to multiple cancers and neurodegenerative disorders, making it a key therapeutic target. However, a detailed understanding of its dynamic functions has been limited by the lack of chemical tools for its visualization in living systems. Utilizing the highly selective inhibitor Nexturastat A as a targeting scaffold, we developed 6SiR-C3-NextA probe incorporating a bright, photostable, far-red silicon-rhodamine fluorophore. Biochemical and cellular assays show that 6SiR-C3-NextA binds to HDAC6 with high affinity (Kdapp = 21 {+/-} 4 nM) and selectivity over other HDAC enzymes, as validated using a developed panel of engineered cell lines expressing individual human HDACs. The probe is cell-permeable, exhibits low cytotoxicity, compatible with super-resolution technique and enables the visualization of endogenous HDAC6 across multiple cell lines. We demonstrate its utility by performing imaging of HDAC6s association with the microtubule network and its dynamic recruitment to stress granules in living cells.

biochemistry↗

bioRxiv2025

Biocompatible sulfonium-based covalent probes for endogenous tubulin fluorescence nanoscopy in live and fixed cells

Fluorescent probes enable the visualization of dynamic cellular processes with high precision, particularly when coupled with super-resolution imaging techniques that surpass the diffraction limit. Traditional methods include fluorescent protein fusion (e.g., GFP) or organic fluorophores linked to ligands targeting the protein of interest. However, these approaches often introduce functional disruptions or ligand-associated biological effects. Herein, we address these challenges by developing covalent fluorescent probes for endogenous tubulin, a critical cytoskeletal protein involved in processes such as cell movement, division, and biomolecule trafficking. Using well-known tubulin binder cabazitaxel and cell permeable fluorophore silicon-rhodamine--as a basis, we introduce a novel biocompatible cleavable linker containing a sulfonium center. This allowed the construction of the optimized probe, 6-SiR-o-C9-CTX, demonstrating excellent cell permeability, fluorogenic properties, and the ability to covalently label tubulin across various human cell lines. Importantly, the targeting moiety could be washed out while preserving tubulin staining, ensuring minimal disruption of tubulin function. This labeling technique is compatible with STED nanoscopy in both live and fixed cells, offering a powerful high-resolution imaging tool.

biochemistry↗

bioRxiv2024

Autonomous bioluminescence emission from transgenic mice

The bacterial bioluminescence system can be applied to produce autonomous bioluminescence in mammalian cells. Until now, the system has only been stably inserted into cells cultured in vitro. Here we report the generation of an autobioluminescent transgenic mouse line constitutively expressing the genes of the bacterial bioluminescence system, enabling substrate-free in vivo luminescence imaging of a living mammal.

bioengineering↗

bioRxiv2022

A general, highly efficient and facile synthesis of biocompatible rhodamine dyes and probes for live-cell multicolor nanoscopy

The development of live-cell fluorescence nanoscopy is powered by the availability of suitable fluorescent probes. Rhodamines are among the best fluorophores for labeling intracellular structures. Isomeric tuning is a powerful method for optimizing biocompatibility of the rhodamine-containing probes without affecting their spectral properties. However, the efficient synthesis pathway for rhodamine 4-isomers is still lacking. Herein, we present a facile protecting-group-free 4-carboxyrhodamines synthesis based on nucleophilic addition of lithium dicarboxybenzenide to the corresponding xanthone. This approach drastically reduces the number of synthesis steps and expands the achievable structural diversity, increases overall yields and permits a cheap gram-scale synthesis of the dyes. We prepared a wide range of symmetric and asymmetric 4-carboxyrhodamines covering the whole visible spectrum and targeted them to multiple structures in living cells - microtubules, DNA, actin, mitochondria, lysosomes, Halo-tagged and SNAP-tagged proteins. The enhanced permeability fluorescent probes operate at submicromolar concentrations allowing high contrast STED and confocal microscopy of living cells and tissues.

biochemistry↗

bioRxiv2021

Blinking fluorescent probes for tubulin nanoscopy in living and fixed cells

Here we report a small molecule probe for single molecule localisation microscopy (SMLM) of tubulin in living and fixed cells. We explored a series of constructs composed of taxanes and spontaneously blinking far-red dye hydroxymethyl silicon-rhodamine (HMSiR). We found that the linker length profoundly affects the probe permeability and off-targeting. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and 6-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameters of [≤]50 nm can be routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin also performs well in 3D stimulated emission depletion (STED) microscopy, allowing a complementary use of both nanoscopy methods for investigating microtubule functions in living cells.

cell biology↗