A5008979377- DNA and Nucleic Acid Chemistry
- Advanced biosensing and bioanalysis techniques
- RNA and protein synthesis mechanisms
- RNA Interference and Gene Delivery
- Advanced NMR Techniques and Applications
- Electron Spin Resonance Studies
- Chronic Myeloid Leukemia Treatments
- Trypanosoma species research and implications
- Genomics and Chromatin Dynamics
- Fibroblast Growth Factor Research
- Protein Structure and Dynamics
- RNA modifications and cancer
- Chromosomal and Genetic Variations
- Viral Infectious Diseases and Gene Expression in Insects
- Epigenetics and DNA Methylation
- Advanced MRI Techniques and Applications
- Wnt/β-catenin signaling in development and cancer
- Virus-based gene therapy research
- Cancer-related gene regulation
- PI3K/AKT/mTOR signaling in cancer
- CAR-T cell therapy research
- Polar Research and Ecology
- Mast cells and histamine
- Biochemical and Molecular Research
- Connective tissue disorders research
Central European Institute of Technology
2016-2025
Central European Institute of Technology – Masaryk University
2016-2025
Czech Academy of Sciences
2003-2024
Czech Academy of Sciences, Institute of Biophysics
2017-2024
Masaryk University
2018-2023
Czech Academy of Sciences, Biology Centre
2007-2012
University Medical Center Utrecht
2012
Utrecht University
2012
Institute of Parasitology
2003-2011
Max Planck Innovation
2009-2011
Abstract C‐rich DNA has the capacity to form a tetra‐stranded structure known as an i‐motif. The i‐motifs within genomic have been proposed contribute regulation of transcription. However, direct experimental evidence for existence these structures in vivo missing. Whether i‐motif complex environment living cells is not currently known. Herein, using state‐of‐the‐art in‐cell NMR spectroscopy, we evaluate stabilities cellular environment. We show that formed from naturally occurring sequences...
Abstract I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites i-motif propensity (iMFPS) human cells, yet their specific contributions remain unclear. Using in-cell NMR oligonucleotide iMFPS models, we monitor iM-associated structural equilibria asynchronous cell cycle-synchronized HeLa cells...
G-quadruplex topologies of telomeric repeat sequences from vertebrates were investigated in the presence molecular crowding (MC) mimetics, namely polyethylene glycol 200 (PEG), Ficoll 70 as well Xenopus laevis egg extract by CD and NMR spectroscopy native PAGE. Here, we show that conformational behavior repeats X. or is notably different observed PEG. While resembles results obtained under dilute conditions, PEG promotes formation high-order parallel topologies. Our data suggest should not...
In-cell NMR spectroscopy of proteins in different cellular environments is a well-established technique that, however, has not been applied to nucleic acids so far. Here, we show that isotopically labeled DNA and RNA can be observed inside the eukaryotic environment Xenopus laevis oocytes by in-cell spectroscopy. One limiting factor for observation their reduced stability. We demonstrate chemical modification protect them from degradation significantly enhance lifetime. Finally, imino region...
Studies on DNA–ligand interactions in the cellular environment are problematic due to lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring inside nuclei living human cells. Our method relies acquisition NMR data from cells electroporated with preformed complexes. The impact intracellular integrity complexes is assessed based signals unbound and ligand-bound forms a given DNA target. This technique was tested two model fragments...
Breast cancer is one of the most common types in women. One genes that were found mutated breast casein kinase 1 epsilon (CK1ε). Because CK1ε a crucial regulator Wnt signaling cascades, we determined how these mutations interfere with pathway and affect behavior epithelial cell lines. We performed silico modeling various analyzed activity mutants both vitro vivo. Furthermore, used reporter small GTPase assays to identify mutation affects different branches pathway. Based on results, employed...
Most of our knowledge protein structure and function originates from experiments performed with purified proteins resuspended in dilute, buffered solutions. However, most crowded intracellular environments complex compositions. Significant efforts have been made to develop tools study their native cellular settings. Among these tools, in-cell NMR spectroscopy has the sole technique for characterizing space living cells at atomic resolution physiological temperature. Nevertheless, due...
Fluorescence resonance energy transfer (FRET) under in vivo conditions is a well-established technique for the evaluation of populations protein bound/unbound nucleic acid (NA) molecules or NA hybridization kinetics. However, FRET has not been applied to quantitative conformational analysis thus far. Here we explored parameters critical characterization structure using single-pair (sp)FRET complex cellular environment living Escherichia coli cell. Our measurements showed that fluorophore...
The blocking of specific protein-protein interactions using nanoparticles is an emerging alternative to small molecule-based therapeutic interventions. However, the designed as “artificial proteins” generally require modification their surface with (bio)organic molecules and/or polymers ensure selectivity and specificity action. Here, we show that nanosized diamond crystals (nanodiamonds, NDs) without any synthetically installed interface enable efficient targeting family extracellular...
Recently, the 1H-detected in-cell NMR spectroscopy has emerged as a unique tool allowing characterization of interactions between nucleic acid-based targets and drug-like molecules in living human cells. Here, we assess application potential 1H 19F-detected to profile drugs/ligands targeting DNA G-quadruplexes, arguably most studied class anti-cancer drugs acids. We show that extension original approach is not straightforward. The severe signal broadening overlap spectra polymorphic...
There are two basic mechanisms that associated with the maintenance of telomere length, which endows cancer cells unlimited proliferative potential. One mechanism, referred to as alternative lengthening telomeres (ALT), accounts for approximately 10–15% all human cancers. Tumours engaged in ALT pathway characterised by presence single stranded 5′-C-rich telomeric overhang (C-overhang). This recently identified hallmark cancers distinguishes them from healthy tissues and renders C-overhang a...
We describe a novel, fundamental property of nucleobase structure, namely, pyramidilization at the N1/9 sites purine and pyrimidine bases. Through combined analyses ultra-high-resolution X-ray structures both oligonucleotides extracted from Nucleic Acid Database isolated nucleotides nucleosides Cambridge Structural Database, together with series quantum chemical calculations, molecular dynamics (MD) simulations, published solution nuclear magnetic resonance (NMR) data, we show that...
Abstract C‐rich DNA has the capacity to form a tetra‐stranded structure known as an i‐motif. The i‐motifs within genomic have been proposed contribute regulation of transcription. However, direct experimental evidence for existence these structures in vivo missing. Whether i‐motif complex environment living cells is not currently known. Herein, using state‐of‐the‐art in‐cell NMR spectroscopy, we evaluate stabilities cellular environment. We show that formed from naturally occurring sequences...
In-cell profiling enables the evaluation of receptor tyrosine activity in a complex environment regulatory networks that affect signal initiation, propagation and feedback. We used FGF-receptor signaling to identify EGR1 as locus strongly responds activation majority recognized protein kinase oncogenes, including 30 kinases 154 their disease-associated mutants. The promoter was engineered enhance trans-activation capacity optimized for simple screening assays with luciferase or fluorescent...
Abstract Metal ions are essential components for the survival of living organisms. For most species, intracellular and extracellular ionic conditions differ significantly. As G-quadruplexes (G4s) ion-dependent structures, changes in [Na+]/[K+] ratio may affect folding genomic G4s. More than 11000 putative G4 sequences human genome (hg19) contain at least two runs three continuous cytosines, these mixed G/C-rich form a quadruplex or competing hairpin structure based on G-C base pairing. In...
ABSTRACT The Sm-binding site of the kinetoplastid spliced leader RNA has been implicated in accurate maturation and trans -splicing competence. In Trypanosoma brucei , interference-mediated knockdown SmD1 caused defects maturation, displaying aberrant 3′-end formation, partial formation cap 4, overaccumulation cytoplasm; U 28 pseudouridylation was unaffected.
AbstractmRNA cap 1 2′-O-ribose methylation is a widespread modification that implicated in processing, trafficking, and translational control eukaryotic systems. The enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) polycistronic precursors conveys hypermethylated 4, including 0 m7G seven additional methylations on the first 4 nucleotides, all nuclear mRNAs. We report methyltransferase, TbMTr1, member conserved family viral enzymes....
In this work, a novel NMR method for the identification of preferential coordination sites between physiologically relevant counterions and nucleic acid bases is demonstrated. approach, cross-correlated relaxation rates aromatic carbon chemical shift anisotropy proton–carbon dipolar interaction are monitored as function increasing Na+, K+, Mg2+ concentrations. Increasing counterion concentration modulates residence times at specific around bases. It demonstrated that modulation leads to...