Introduction Of Fluorophores Research Articles

Fluorescent Covalent Organic Frameworks: A Promising Material Platform for Explosive Sensing.

Covalent organic frameworks (COFs) are a novel class of porous crystalline organic materials with organic small molecule units connected by strong covalent bonds and extending in two- or three-dimension in an ordered mode. The tunability, porosity, and crystallinity have endowed covalent organic frameworks the capability of multi-faceted functionality. Introduction of fluorophores into their backbones or side-chains creates emissive covalent organic frameworks. Compared with common fluorescent organic solid materials, COFs possess several intrinsic advantages being as a type of irreplaceable fluorescence materials mainly because its highly developed pore structures can accommodate various types of guest analytes by specific or non-specific chemical bonding and non-bonding interaction. Developments in fluorescent COFs have provided opportunities to enhance sensing performance. Moreover, due to its inherent rigidified structures and fixed conformations, the intramolecular rotation, vibration, and motion occurred in common organic small molecules, and organic solid systems can be greatly inhibited. This inhibition decreases the decay of excited-state energy as heat and blocks the non-radiative quenching channel. Thus, fluorescent COFs can be designed, synthesized, and precisely tuned to exhibit optimal luminescence properties in comparison with common homogeneous dissolved organic small molecule dyes and can even compete with the currently mainstream organic solid semiconductor-based luminescence materials. This mini-review discusses the major design principle and the state-of-the-art paragon examples of fluorescent COFs and their typical applications in the detection and monitoring of some key explosive chemicals by fluorescence analysis. The challenges and the future direction of fluorescent COFs are also covered in detail in the concluding section.

Site-Specific Fluorescent Labeling of RNA Interior Positions.

The introduction of fluorophores into RNA for both in vitro and in cellulo studies of RNA function and cellular distribution is a subject of great current interest. Here I briefly review methods, some well-established and others newly developed, which have been successfully exploited to site-specifically fluorescently label interior positions of RNAs, as a guide to investigators seeking to apply this approach to their studies. Most of these methods can be applied directly to intact RNAs, including (1) the exploitation of natural posttranslational modifications, (2) the repurposing of enzymatic transferase reactions, and (3) the nucleic acid-assisted labeling of intact RNAs. In addition, several methods are described in which specifically labeled RNAs are prepared de novo.

The Role of Cysteine Residues in the Interaction of Nicking Endonuclease BspD6I with DNA

Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only one strand. The mechanism of their action is important to study because NEs with new specificities are necessary to design to solve the practical tasks of biotechnology. One of the modern approaches for investigation of protein-nucleic acid interactions is fluorescence spectroscopy, which involves the introduction of fluorophores into proteins, mainly through Cys residues due to the high reactivity of their thiol group. To implement this approach, it is necessary to clarify the role of Cys residues in the functioning of the native protein and the possible consequences of their modification. Crosslinking was used to study whether Cys residues are close to DNA in the complex with NE BspD6I. Reactions were carried out using the wild-type enzyme, its mutant form NE BspD6I(C11S/C160S), and modified DNA duplexes containing the 2-pyridyldisulfide group at the C2' atom of the sugar-phosphate moiety in different positions of the oligonucleotide strand. The Cys residues of NE BspD6I were for the first time shown to be in close proximity to DNA during the binding process, including the step of a nonspecific complex formation. The substitutions C11S and C160S in the N-terminal domain of the enzyme slightly decreased the efficiency of substrate hydrolysis. Construction of a cysteine-free NE BspD6I variant and examination of its properties will provide additional information about the functional significance of the Cys residues for this unique enzyme.

Synthesis, Photophysics, and Solvatochromic Studies of an Aggregated-Induced-Emission Luminogen Useful in Bioimaging.

Biological samples are a complex and heterogeneous matrix where different macromolecules with different physicochemical parameters cohabit in reduced spaces. The introduction of fluorophores into these samples, such as in the interior of cells, can produce changes in the fluorescence emission properties of these dyes, caused by the specific physicochemical properties of cells. This effect can be especially intense with solvatofluorochromic dyes, where changes in the polarity environment surrounding the dye can drastically change the fluorescence emission. In this article, we studied the photophysical behavior of a new dye and confirmed the aggregation-induced emission (AIE) phenomenon with different approaches, such as by using different solvent proportions, increasing the viscosity, forming micelles, and adding bovine serum albumin (BSA), through analysis of the absorption and steady-state and time-resolved fluorescence. Our results show the preferences of the dye for nonpolar media, exhibiting AIE under specific conditions through immobilization. Additionally, this approach offers the possibility of easily determining the critical micelle concentration (CMC). Finally, we studied the rate of spontaneous incorporation of the dye into cells by fluorescence lifetime imaging and observed the intracellular pattern produced by the AIE. Interestingly, different intracellular compartments present strong differences in fluorescence intensity and fluorescence lifetime. We used this difference to isolate different intracellular regions to selectively study these regions. Interestingly, the fluorescence lifetime shows a strong difference in different intracellular compartments, facilitating selective isolation for a detailed study of specific organelles.

A Step Forward in the Design of Liposomes: Symmetric & Asymmetric Vesicles from Lipid Extracts

Nature's complexity in the design of cellular boundaries represent a challenge in monitoring processes that are located on or associated with the membrane. Liposomes are the method of choice when studying membrane-peptide or -protein interactions due to the good control regarding composition and the reduced number of component variables. One major drawback in their design is the symmetric architecture and the related lack of controlling different lipid compositions between inner (IL) and outer leaflet (OL). Since biological membranes are highly asymmetric, we compare different techniques for engineering lipid vesicles and point out challenges, opportunities and potential limitations: Symmetric vesicles are prepared by electroformation whereas asymmetric vesicles are generated from a double-emulsion phase. The different fashioned vesicles are either reconstituted from pure DOPC or lipid mixtures such as DOPC:SM:Chol as host cell mimicry and model for lipid rafts as well as POPE:POPG as inner membrane model of Gram-negative bacteria. An extension from these synthetic lipid species to natural lipid extracts, e.g. S. cerevisiae, C. albicans, E. coli, and bovine liver and heart build a bridge between the pure model and native cellular systems. Asymmetric vesicles provide a perfect tool in modelling the bacterial outer leaflet by using lipopolysaccharides (LPS) as OL-mimicry and a phospholipid mixture composed of PE:PG:CL for the IL. Both vesicle design concepts are compared regarding size distribution, reconstitution potential in terms of the applied lipid spectrum, introduction of fluorophores, the ability to undergo phase separation and the dominating domain-sizes as investigated by fluorescence and confocal microscopy. Additionally, asymmetric vesicles are used for the determination of lipid flip-flop with respect to the OL-composition. To which extent lipid specificity and interleaflet coupling play a role in phase separation and lipid flip-flop should be elucidated.

The N-terminal region of two-pore channel 1 regulates trafficking and activation by NAADP

TPCs (two-pore channels) are NAADP (nicotinic acid-adenine dinucleotide phosphate)-sensitive Ca2+-permeable ion channels expressed on acidic organelles. In the present study we show that deletion of the N-terminal region redirects TPC1 to the ER (endoplasmic reticulum). The introduction of fluorophores at the N-terminus of TPC1 does not affect its subcellular location, but does reversibly abolish NAADP sensitivity. Our results reveal a dual role for the N-terminus in localization and function of TPC1.

Label-free detection of drug-membrane association using ultraviolet-visible sum-frequency generation.

Drug-membrane interactions play a crucial role in the pharmacology and activity of drugs. The measurement of drug association to lipid membranes has conventionally been measured by fluorescence and other spectroscopic methods. However, a main disadvantage of fluorescence labeling of drugs is that the introduction of fluorophores may change the molecules physical properties, such as charge, hydrophobic or hydrophilic character, and structure. To circumvent these problems, Ultraviolet-Visible Sum Frequency Generation (UV-Vis SFG) has been developed as an ultrasensitive and label-free technique to detect small-molecule drug association to lipid membranes. Four different classes of drugs, a nonsteroidal anti-inflammatory drug (ibuprofen), antibiotic (azithromycin), antifungal (tolnaftate), and local anesthetic (tetracaine), were examined. Drug association was measured on planar supported lipid bilayers (PSLBs) of 1,2-dioleoyl-sn-glycero-3-phophocholine (DOPC). Equilibrium association constants of the drugs were obtained and correlate well to the partition coefficients of the drugs in a liposome membrane-water system. UV-Vis SFG is a powerful and novel technique to directly measure the association of drugs to a single biological membrane without chemical modification.

Determination of DNA-binding parameters for the Bacillus subtilis histone-like HBsu protein through introduction of fluorophores by site-directed mutagenesis of a synthetic gene.

A synthetic gene encoding the histone-like DNA-binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli. Yields of the purified protein are at least 20 mg/l culture medium. The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild-type protein. N-terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E. coli. Site-directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA-binding parameters by fluorescence spectroscopy. According to gel-retardation experiments, the mutant protein [Phe47----Trp]HBsu shows identical DNA binding to wild-type HBsu protein. Analysis of fluorescence binding data reveals that [Phe47----Trp]HBsu binds double-stranded DNA with a dissociation constant in the micromolar range. Computer-assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely. A dimer of [Phe47----Trp]HBsu appears to contact three or four base pairs of DNA. These results are in partial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.