Biology subjects

Gerasimaite, R.

Publications and source records attributed to Gerasimaite, R..

6 recordsLinked to original sources

bioRxiv2026

Near-Infrared Turn-On Fluorogenic Probe for Versatile Detection of Inorganic Polyphosphates

Inorganic polyphosphate (polyP) is a ubiquitous phosphate biopolymer involved in diverse cellular processes. Despite its significance, selective detection of polyP remains challenging because of its simple and highly charged structure. Here, we report a near-infrared (NIR) fluorogenic turn-on chemosensor for selective polyP detection and imaging, SiX-DPA-Zn. The probe combines a silicon-xanthene (SiX) fluorophore with a zinc(II)-coordinated 2,2'-dipicolylamine (DPA-Zn2+) recognition unit and shows more than 100-fold selectivity for inorganic polyP over ADP and ATP. SiX-DPA-Zn enables quantitative detection of polyP at micromolar concentrations in microplate assays and stains a broad range of polyP species, starting from tripolyphosphate, in polyacrylamide gels. In HEK293 cells expressing Escherichia coli polyphosphate kinase 1, the probe visualizes intracellular polyP and enables quantitative analysis of polyP levels in relation to nuclear proteins for example fibrillarin and nucleolin. Stimulated emission depletion (STED) microscopy further revealed subdiffraction-sized polyP granules within polyP aggregates. SiX-DPA-Zn is the first near-infrared (NIR) fluorogenic chemosensor for polyP that is compatible with multiple detection platforms, including microplate assays, polyacrylamide gel staining, confocal and super-resolution STED microscopy.

biochemistry↗

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↗

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

Overcoming efflux of fluorescent probes for actin imaging in living cells

Actin cytoskeleton is crucial for endocytosis, intracellular trafficking, cell shape maintenance and a wide range of other cellular functions. Recently introduced cell-permeable fluorescent actin probes suffer from poor membrane permeability and stain some cell populations inhomogeneously due to the active efflux by the plasma membrane pumps. We addressed this issue by constructing a series of probes which employ modified rhodamine fluorophores. We found that the best performing probes are based on 6-carboxy-carbopyronine scaffold. These probes show preferential binding to F-actin, do not require efflux pumps inhibitors for staining and can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity. We demonstrate their excellent performance in multiple organisms and cell types: human cell lines, frog erythrocytes, fruit fly tissues and primary neurons.

biochemistry↗