Biology subjects

Bucevicius, J.

Publications and source records attributed to Bucevicius, J..

8 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↗

bioRxiv2026

Dual Stabilization of S-Adenosylmethionine for Enzymatic DNA Labeling

S-Adenosyl-L-methionine (AdoMet) analogues are powerful tool for site-specific biomolecular labeling via methyltransferase (MTase) catalyzed transfer reactions. However, their utility is often limited by poor chemical stability under enzymatic reaction conditions. Here, we report a new class of stabilized AdoMet analogues featuring a conformationally constrained proline side chain in place of homoalanine. This substitution inhibits intramolecular cyclization, a major decomposition pathway. Combination with selenonium modification, which suppresses depurination, yields analogues with up to a 90-fold increase half-life relative to AdoMet. These cofactors retain activity with DNA MTases, and allow sequence-specific labeling of plasmid DNA using both two-step and single-step approaches with fluorescent dyes.

bioengineering↗

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↗

bioRxiv2022

Biophysical ordering transitions underlie genome 3D re-organization during cricket spermiogenesis.

Spermiogenesis is a radical process of differentiation whereby sperm cells acquire a compact and specialized morphology to cope with the constraints of sexual reproduction while preserving their main cargo, an intact copy of the paternal genome. In animals, this often involves the replacement of most histones by sperm-specific nuclear basic proteins (SNBPs). Yet, how the SNBP-structured genome achieves compaction and accommodates shaping remain largely unknown. Here, we exploited confocal, electron and super-resolution microscopy observations, coupled with polymer modeling simulations to identify the higher-order architecture of sperm chromatin in the needle-shaped nucleus of the emerging model cricket Gryllus bimaculatus. Accompanying spermatid differentiation and shaping, the SNBP-based genome was strikingly reorganized as ~25nm-thick fibers orderly coiled along the elongated nucleus axis. This chromatin spool was further found to achieve large-scale helical twisting in the final stages of spermiogenesis, favoring its ultracompaction. Through a combination of microscopy observations and polymer simulations, we revealed that these dramatic transitions may be recapitulated by a surprisingly simple biophysical principle based on a nucleated rigidification of chromatin linked to the histone-to-SNBP transition within a confined nuclear space. Our work highlights a unique, liquid crystal-like mode of higher-order genome organization in ultracompact cricket sperm completely distinct from nucleosomal chromatin, and establishes a multidisciplinary methodological framework to explore the diversity of non-canonical modes of DNA organization. SIGNIFICANCE STATEMENTAnimal sperm cells are highly compact and atypically shaped compared to other cell types. How DNA is packaged and organized in the 3D space of sperm cell nuclei to cope with these constraints is poorly understood. In this work, we identified an original and elegant solution to this problem in crickets, whereby DNA fibers orderly spool and twist to fit into ultracompact, needle-shaped sperm cells. To understand this reorganization, we modeled DNA fibers in the nucleus as polymers and found that a relatively simple mechanism through which fibers become more rigid bit by bit can largely recapitulate our observations. Our multidisciplinary work highlights a simple solution to compact DNA to extreme levels in specialized nuclei.

cell biology↗

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↗

bioRxiv2020

Enhancing biocompatibility of rhodamine fluorescent probes by a neighbouring group effect

Fluorescence microscopy is an essential tool for understanding dynamic processes in living cells and organisms. However, many fluorescent probes for labelling cellular structures suffer from unspecific interactions and low cell permeability. Herein, we demonstrate that the neighbouring group effect which results from positioning an amide group next to a carboxyl group in the benzene ring of rhodamines dramatically increases cell permeability of the rhodamine-based probes through stabilizing a fluorophore in a hydrophobic spirolactone state. Based on this principle, we create probes targeting tubulin, actin and DNA. Their superb staining intensity, tuned toxicity and specificity allows long-term 3D confocal and STED nanoscopy with sub-30 nm resolution. As a result, the real microtubule diameter of 23 nm was resolved inside a living cell for the first time. Due to their unrestricted cell permeability and efficient accumulation on tubulin, the new probes produce high contrast images at sub-nanomolar concentrations.

biochemistry↗