Optical Mapping: Detecting Genomic Resistance Cassettes in MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium with a global presence in healthcare facilities as well as community settings. The resistance of MRSA to beta-lactam antibiotics can be attributed to a mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec), ranging from 23 to 68 kilobase pairs in length. The mec gene complex contained in SCCmec allows MRSA to survive in the presence of penicillin and other beta-lactam antibiotics. We demonstrate that optical mapping (OM) is able to identify the bacterium as S. aureus, followed by an investigation of the presence of kilobase pair range SCCmec elements by examining the associated OM-generated barcode patterns. By employing OM as an alternative to traditional DNA sequencing, we showcase its potential for the detection of complex genetic elements such as SCCmec in MRSA. This approach holds promise for enhancing our understanding of antibiotic resistance mechanisms and facilitating the development of targeted interventions against MRSA infections.

Trifunctional Linkers Enable Improved Visualization of Actin by Expansion Microscopy.

Expansion microscopy (ExM) revolutionized the field of super-resolution microscopy by allowing for subdiffraction resolution fluorescence imaging on standard fluorescence microscopes. However, it has been found that it is hard to visualize actin filaments efficiently using ExM. To improve actin imaging, multifunctional molecules have been designed with moderate success. Here, we present optimized methods for phalloidin conjugate grafting that have a high efficiency for both cellular and tissue samples. Our optimized strategy improves anchoring and signal retention by ∼10 times. We demonstrate the potential of optimized trifunctional linkers (TRITON) for actin imaging in combination with immunolabeling using different ExM protocols. 10X ExM of actin labeled with optimized TRITON enabled us to visualize the periodicity of actin rings in cultured hippocampal neurons and brain slices by Airyscan confocal microscopy. Thus, TRITON linkers provide an efficient grafting method, especially in cases in which the concentration of target-bound monomers is insufficient for high-quality ExM.

Current Progress in Expansion Microscopy: Chemical Strategies and Applications.

Expansion microscopy (ExM) is a newly developed super-resolution technique, allowing visualization of biological targets at nanoscale resolution on conventional fluorescence microscopes. Since its introduction in 2015, many efforts have been dedicated to broaden its application range or increase the resolution that can be achieved. As a consequence, recent years have witnessed remarkable advances in ExM. This review summarizes recent progress in ExM, with the focus on the chemical aspects of the method, from chemistries for biomolecule grafting to polymer synthesis and the impact on biological analysis. The combination of ExM with other microscopy techniques, in search of additional resolution improvement, is also discussed. In addition, we compare pre- and postexpansion labeling strategies and discuss the impact of fixation methods on ultrastructure preservation. We conclude this review with a perspective on existing challenges and future directions. We believe that this review will provide a comprehensive understanding of ExM and facilitate its usage and further development.

Improved Dye Survival in Expansion Microscopy through Stabilizer-Conjugated Linkers.

Expansion microscopy (ExM) has been widely used to detect biomolecules in cultured cells and tissue samples due to its enablement of super resolution imaging with conventional microscopes, via physical expansion of samples. However, reaction conditions inherent to the process bring about strong fluorescent signal loss during polymerization and digestion and thus limit the brightness of the signal obtained post expansion. Here, we explore the impact of stabilizer-containing organic fluorophores in ExM, as a mitigation strategy for this radical-induced dye degradation. Through direct conjugation of 4-nitrophenylalanine (NPA) to our previously developed trifunctional reagents, we validate and demonstrate that these multifunctional linkers enable visualization of different organelles with improved fluorescent intensity, owning to protection of the dyes to radical induced degradation as well as to photoprotection upon imaging. At this point, we cannot disentangle the relative contribution of both mechanisms. Furthermore, we report anchoring linkers that allow straightforward application of NPA or Trolox to commercially available fluorophore-conjugated antibodies. We show that these anchoring linkers enable complete retention of biological targets while increasing fluorophore photostability. Our results provide guidance in exploring these stabilizer-modified agents in ExM and methods for increased signal survival through the polymerization steps of the ExM protocols.

A Universal Labeling Strategy for Nucleic Acids in Expansion Microscopy.

Expansion microscopy (ExM) enables the nanoscale imaging of ribonucleic acids (RNAs) on a conventional fluorescence microscope, providing information on the intricate patterns of gene expression at (sub)cellular resolution and within spatial context. To extend the use of such strategies, we examined a series of multivalent reagents that allow the labeling and grafting of deoxyribonucleic acid (DNA) oligonucleotide probes in a unified approach. We show that the reagents are directly compatible with third-generation in situ hybridization chain reaction RNA FISH (fluorescence in situ hybridization) techniques while displaying complete retention of the targeted transcripts. Furthermore, we validate and demonstrate that our labeling method is compatible with multicolor staining. Through oligonucleotide-conjugated antibodies, we demonstrate excellent performance in ×4 ExM and ×10 ExM, achieving a resolution of ∼50 nm in ×10 ExM for both pre- and postexpansion labeling strategies. Our results indicate that our multivalent molecules enable the rapid functionalization of DNA oligonucleotides for ExM.

Evaluation of Direct Grafting Strategies via Trivalent Anchoring for Enabling Lipid Membrane and Cytoskeleton Staining in Expansion Microscopy.

Super-resolution fluorescence microscopy is a key tool in the elucidation of biological fine structures, providing insights into the distribution and interactions of biomolecular complexes down to the nanometer scale. Expansion microscopy is a recently developed approach for achieving nanoscale resolution on a conventional microscope. Here, biological samples are embedded in an isotropically swollen hydrogel. This physical expansion of the sample allows imaging with resolutions down to the tens-of-nanometers. However, because of the requirement that fluorescent labels are covalently bound to the hydrogel, standard, small-molecule targeting of fluorophores has proven incompatible with expansion microscopy. Here, we show a chemical linking approach that enables direct, covalent grafting of a targeting molecule and fluorophore to the hydrogel in expansion microscopy. We show application of this series of molecules in the antibody-free targeting of the cell cytoskeleton and in an example of lipid membrane staining for expansion microscopy. Furthermore, using this trivalent linker strategy, we demonstrate the benefit of introducing fluorescent labels post-expansion by visualizing an immunostaining through fluorescent oligonucleotide hybridization after expanding the polymer. Our probes allow different labeling approaches that are compatible with expansion microscopy.

Fluorescent SAM analogues for methyltransferase based DNA labeling.

In this work, the preparation of new S-adenosyl-l-methionine (SAM) analogues for sequence specific DNA labeling is evaluated. These non-natural analogues, comprising cysteine rather than the natural homolog, were obtained in near quantitative conversions from readily available starting materials without relying on using an excess amount of labor intensive molecules. The synthetic strategy was used to generate fluorescent cofactors, with colours spanning the whole visible spectrum, and their applicability in methyltransferase based optical mapping is shown.

Impact of the Keto-Enol Tautomeric Equilibrium on the BODIPY Chromophore.

An intramolecular tautomeric fluorescent BODIPY sensor has been designed and synthesized. The obtained BODIPY dye is a combination of the 4-bora- 3a, 4a-diaza- s-indacene core and a diketone fragment. The study of conformational equilibria in the ground and excited states has been completed for a broad range of solvent polarity by steady state and NMR methods as well as by DFT and TD-DFT calculations. The interpretation of the unique emission observed in hydrogen bond accepting solvents upon the excitation of the fluorescent dye in the S0-S2 transition has been accomplished. The Jablonski diagram has been analyzed for the observed processes in the BODIPY dye studied on the basis of DFT and TD-DFT calculations.

A general strategy for direct, enzyme-catalyzed conjugation of functional compounds to DNA.

The methyltransferase enzymes can be applied to deliver a range of modifications to pre-determined sites on large DNA molecules with exceptional specificity and efficiency. To date, however, a limited number of modifications have been delivered in this way because of the complex chemical synthesis that is needed to produce a cofactor analogue carrying a specific function, such as a fluorophore. Here, we describe a method for the direct transfer of a series of functional compounds (seven fluorescent dyes, biotin and polyethylene glycol) to the DNA duplex. Our approach uses a functional cofactor analogue, whose final preparative step is performed alongiside the DNA modification reaction in a single pot, with no purification needed. We show that fluorophore conjugation efficiency in these mixtures is significantly improved compared to two-step labeling approaches. Our experiments highlight the remarkable malleability and selectivity of the methyltransferases tested. Additional analysis using high resolution localization of the fluorophore distribution indicates that target sites for the methyltransferase are predominantly labeled on a single strand of their palindromic site and that a small and randomly-distributed probability of off-site labeling exists.

Methyltransferase-directed covalent coupling of fluorophores to DNA.

We report an assay for determining the number of fluorophores conjugated to single plasmid DNA molecules and apply this to compare the efficiency of fluorophore coupling strategies for covalent DNA labelling. We compare a copper-catalyzed azide-alkyne cycloaddition reaction, amine to N-hydroxysuccinimidyl ester coupling reaction and strain-promoted azide-alkyne cycloaddition reaction for fluorescent DNA labelling. We found increased labelling efficiency going from the amine to N-hydroxysuccinimidyl ester coupling reaction to the copper-catalyzed azide-alkyne cycloaddition and found the highest degree of DNA labelling with the strain-promoted azide-alkyne cycloaddition reaction. We also examined the effect of labelling on the DNA structure using atomic force microscopy. We observe no distortions or damage to the DNA that was labeled using the amine to N-hydroxysuccinimidyl ester and strain-promoted azide-alkyne cycloaddition coupling reactions. This was in contrast to the copper-catalyzed azide-alkyne cycloaddition reaction, which, despite the use of copper-coordinating ligands in the labelling mixture, leads to some structural DNA damage (single-stranded DNA breaks).

Methyltransferase-Directed Labeling of Biomolecules and its Applications.

Methyltransferases (MTases) form a large family of enzymes that methylate a diverse set of targets, ranging from the three major biopolymers to small molecules. Most of these MTases use the cofactor S-adenosyl-l-Methionine (AdoMet) as a methyl source. In recent years, there have been significant efforts toward the development of AdoMet analogues with the aim of transferring moieties other than simple methyl groups. Two major classes of AdoMet analogues currently exist: doubly-activated molecules and aziridine based molecules, each of which employs a different approach to achieve transalkylation rather than transmethylation. In this review, we discuss the various strategies for labelling and functionalizing biomolecules using AdoMet-dependent MTases and AdoMet analogues. We cover the synthetic routes to AdoMet analogues, their stability in biological environments and their application in transalkylation reactions. Finally, some perspectives are presented for the potential use of AdoMet analogues in biology research, (epi)genetics and nanotechnology.

A Blue-Light-Emitting BODIPY Probe for Lipid Membranes.

Here we describe a new BODIPY-based membrane probe (1) that provides an alternative to dialkylcarbocyanine dyes, such as DiI-C18, that can be excited in the blue spectral region. Compound 1 has unbranched octadecyl chains at the 3,5-positions and a meso-amino function. In organic solvents, the absorption and emission maxima of 1 are determined mainly by solvent acidity and dipolarity. The fluorescence quantum yield is high and reaches 0.93 in 2-propanol. The fluorescence decays are well fitted with a single-exponential in pure solvents and in small and giant unilamellar vesicles (GUV) with a lifetime of ca. 4 ns. Probe 1 partitions in the same lipid phase as DiI-C18(5) for lipid mixtures containing sphingomyelin and for binary mixtures of dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC). The lipid phase has no effect on the fluorescence lifetime but influences the fluorescence anisotropy. The translational diffusion coefficients of 1 in GUVs and OLN-93 cells are of the same order as those reported for DiI-C18. The directions of the absorption and emission transition dipole moments of 1 are calculated to be parallel. This is reflected in the high steady-state fluorescence anisotropy of 1 in high ordered lipid phases. Molecular dynamic simulations of 1 in a model of the DOPC bilayer indicate that the average angle of the transition moments with respect to membrane normal is ca. 70°, which is comparable with the value reported for DiI-C18.

Improved Spectral Coverage and Fluorescence Quenching in Donor-acceptor Systems Involving Indolo[3-2-b]carbazole and Boron-dipyrromethene or Diketopyrrolopyrrole.

A novel π-conjugated triad and a polymer incorporating indolo[3,2-b]-carbazole (ICZ) and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) were synthesized via a Sonogashira coupling. Compared to the parent BODIPY the absorption and fluorescence spectrum were for both compounds broader and redshifted. The redshift of the fluorescence and the decrease of the fluorescence quantum yield and decay time upon increasing solvent polarity were attributed to the formation of a partial charge-transfer state. Upon excitation in the ICZ absorption band the ICZ fluorescence was quenched in both compounds mainly due to energy transfer to the BODIPY moiety. In a similar ICZ-π-DPP polymer (where DPP is diketopyrrolopyrrole), a smaller redshift of the absorption and fluorescence spectra compared to the parent DPP was observed. A less efficient quenching of the ICZ fluorescence in the ICZ-π-DPP polymer could be related to the unfavorable orientation of the transition dipoles of ICZ and DPP. The rate constant for energy transfer was for all compounds an order of magnitude smaller than predicted by Förster theory. While in a solid film of the triad a further redshift of the absorption maximum of nearly 100 nm was observed, no such shift was observed for the ICZ-π-BODIPY polymer.

Solvatochromism of BODIPY-Schiff dye.

A boron-dipyrrin chromophore connected with an o-hydroxyaryl aldimine by a diazo bridge (BODIPY-Schiff dye) has been developed. The photophysical properties of the BODIPY-Schiff dye have been investigated with UV, steady-state, and time-resolved fluorimetry. The spectral features have been characterized with respect to density functional theory and time-dependent density functional theory. The conformational analysis of the studied compound has been accomplished both in the ground and excited states. A scheme of the processes occurring in the BODIPY-Schiff dye has been proposed.

8-HaloBODIPYs and their 8-(C, N, O, S) substituted analogues: solvent dependent UV-vis spectroscopy, variable temperature NMR, crystal structure determination, and quantum chemical calculations.

The UV-vis electronic absorption and fluorescence emission properties of 8-halogenated (Cl, Br, I) difluoroboron dipyrrin (or 8-haloBODIPY) dyes and their 8-(C, N, O, S) substituted analogues are reported. The nature of the meso-substituent has a significant influence on the spectral band positions, the fluorescence quantum yields, and lifetimes. As a function of the solvent, the spectral maxima of all the investigated dyes are located within a limited wavelength range. The spectra of 8-haloBODIPYs display the narrow absorption and fluorescence emission bands and the generally quite small Stokes shifts characteristic of classic difluoroboron dipyrrins. Conversely, fluorophores with 8-phenylamino (7), 8-benzylamino (8), 8-methoxy (9), and 8-phenoxy (10) groups emit in the blue range of the visible spectrum and generally have larger Stokes shifts than common BODIPYs, whereas 8-(2-phenylethynyl)BODIPY (6) has red-shifted spectra compared to ordinary BODIPY dyes. Fluorescence lifetimes for 6, 8, and 10 have been measured for a large set of solvents and the solvent effect on their absorption and emission maxima has been analyzed using the generalized Catalán solvent scales. Restricted rotation about the C8-N bond in 7 and 8 has been observed via temperature dependent (1)H NMR spectroscopy, whereas for 10 the rotation about the C8-O bond is not hindered. The crystal structure of 8 demonstrates that the short C8-N bond has a significant double character and that this N atom exhibits a trigonal planar geometry. The crystal structure of 10 shows a short C8-O bond and an intramolecular C-H···π interaction. Quantum-chemical calculations have been performed to assess the effect of the meso-substituent on the spectroscopic properties.

Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry.

We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes transalkylate DNA with the cofactor we tested (a readily prepared s-adenosyl-l-methionine analogue).

Design and synthesis of nucleolipids as possible activated precursors for oligomer formation via intramolecular catalysis: stability study and supramolecular organization.

BACKGROUND: Fatty acid vesicles are an important part of protocell models currently studied. As protocells can be considered as pre-biological precursors of cells, the models try to contribute to a better understanding of the (cellular) origin of life and emphasize on 2 major aspects: compartmentalization and replication. It has been demonstrated that lipid-based membranes are amenable to growth and division (shell replication). Furthermore compartmentalization creates a unique micro-environment in which biomolecules can accumulate and reactions can occur. Pioneering research by Sugawara, Deamer, Luisi, Szostak and Rasmussen gave more insight in obtaining autocatalytic, self-replicating vesicles capable of containing and reproducing nucleic acid sequences (core replication). Linking both core and shell replication is a challenging feat requiring thorough understanding of membrane dynamics and (auto)catalytic systems. A possible solution may lie in a class of compounds called nucleolipids, who combine a nucleoside, nucleotide or nucleobase with a lipophilic moiety. Early contributions by the group of Yanagawa mentions the prebiotic significance (as a primitive helical template) arising from the supramolecular organization of these compounds. Further contributions, exploring the supramolecular scope regarding phospoliponucleosides (e.g. 5'-dioleylphosphatidyl derivatives of adenosine, uridine and cytidine) can be accounted to Baglioni, Luisi and Berti. This emerging field of amphiphiles is being investigated for surface behavior, supramolecular assembly and even drug ability. RESULTS: A series of α/ß-hydroxy fatty acids and α-amino fatty acids, covalently bound to nucleoside-5'-monophosphates via a hydroxyl or amino group on the fatty acid was examined for spontaneous self-assembly in spherical aggregates and their stability towards intramolecular cleavage. Staining the resulting hydrophobic aggregates with BODIPY-dyes followed by fluorescent microscopy gave several distinct images of vesicles varying from small, isolated spheres to higher order aggregates and large, multimicrometer sized particles. Other observations include rod-like vesicle precursors. NMR was used to assess the stability of a representative sample of nucleolipids. 1D 31P NMR revealed that ß-hydroxy fatty acids containing nucleotides were pH-stable while the α-analogs are acid labile. Degradation products identified by [1H-31P] heteroTOCSY revealed that phosphoesters are cleaved between sugar and phosphate, while phosphoramidates are also cleaved at the lipid-phosphate bond. For the latter compounds, the ratio between both degradation pathways is influenced by the nucleobase moiety. However no oligomerization of nucleotides was observed; nor the formation of 3'-5'-cyclic nucleotides, possible intermediates for oligonucleotide synthesis. CONCLUSIONS: The nucleolipids with a deoxyribose sugar moiety form small or large vesicles, rod-like structures, vesicle aggregates or large vesicles. Some of these aggregates can be considered as intermediate forms in vesicle formation or division. However, we could not observe nucleotide polymerization or cyclic nucleotide function of these nucleolipids, regardless of the sugar moiety that is investigated (deoxyribose, ribose, xylose). To unravel this observation, the chemical stability of the constructs was studied. While the nucleolipids containing ß-hydroxy fatty acids are stable as well in base as in acid circumstances, others degraded in acidic conditions. Phosphoramidate nucleolipids hydrolyzed by P-N as well as P-O bond cleavage where the ratio between both pathways depends on the nucleobase. Diester constructs with an α-hydroxy stearic acid degraded exclusively by hydrolysis of the 5'-O-nucleoside ester bond. As the compounds are too stable and harsh conditions would destruct the material itself, more reactive species such as lipid imidazolates of nucleotides need to be synthesized to further analyze the potential polymerization process. Graphical AbstractVesicle information of a nucleolipid consisting of a nucleoside 5'-monophosphate and a α-hydroxy fatty acid.

Synthesis and in vitro evaluation of a PDT active BODIPY-NLS conjugate.

Two new photosensitizers based on the BODIPY scaffold have been synthesized, of which one bears an NLS peptide, which is linked to the BODIPY's core using the copper catalysed azide-alkyne click reaction. The phototoxicities of these BODIPY based photosensitizers have been determined, as well as their dark toxicities. Although the conjugation of a single NLS peptide to the BODIPY did not lead to any observable nuclear localization, the photosensitizer did exhibit a superior photoxicity. Cellular co-localization experiments revealed a localization of both dyes in the lysosomes, as well as a partial localization within the ER (for the peptide-bearing BODIPY).

UV-vis spectroscopy of the coupling products of the palladium-catalyzed C-H arylation of the BODIPY core.

The steady-state, UV-vis electronic absorption and fluorescence emission properties of a large set of 3-aryl and 3,5-diaryl substituted difluoroboron dipyrromethene dyes obtained via direct, palladium-catalyzed C-H (het)arylation of the BODIPY core are reported. The spectra display the narrow absorption and fluorescence emission bands and the generally quite small Stokes shifts characteristic of classic difluoroboron dipyrrins. As a function of the solvent, the spectral maxima are located within a very narrow wavelength range and are slightly red-shifted with increasing solvent polarizability, which is shown to be the crucial parameter influencing the wavelength position of the maxima. The extended π-conjugation in the 3,5-diaryl products always leads to bathochromically shifted absorption and emission spectra compared to those of the 3-aryl analogues. The derivative with a 3-mesityl substituent has blue-shifted spectra in comparison to its 3-phenyl substituted analogue, reflecting the diminished π-conjugation in the former due to steric strain. The nature of the meso-aryl has only a small effect on the spectral positions but affects the fluorescence quantum yield Φ. The majority of the dyes have high Φ (>0.85), except the compounds with meso-phenyl and meso-(p-nitrophenyl) substituents. Quantum-chemical calculations were performed to evaluate the differences in spectroscopic properties upon substitution of the BODIPY core and to compare them with the corresponding experimental results.

Synthesis of meso-halogenated BODIPYs and access to meso-substituted analogues.

8-Halogenated boradiaza-s-indacenes can be efficiently prepared from dipyrrylketones. The new dyes react smoothly with nucleophiles to yield N-, O-, and S-substituted chromophores, as well as transition-metal-catalyzed cross-coupling reactions. The nature of the new substitutent has a strong influence on the spectral properties of the dyes.