Fluorescent Modifications Research Articles

Monitoring Enzymatic RNA G-Quadruplex Unwinding Activities by Nuclease Sensitivity and Reverse Transcription Stop Assays.

Multiple different methods have been employed to investigate the unwinding of RNA G-quadruplexes by various helicase proteins. Each has their own pitfalls, namely, looking at non-native or chemically modified RNA sequences, biasing the unwinding process with competing trap nucleotides, and a lack of context sequence to the 5' and 3' of the RNA G-quadruplex structure. Herein we present two straightforward methods that allow for quadruplex unwinding to be monitored on native RNA sequences without the use of fluorescent modifications, specialized equipment, or trap nucleotides to be employed.

(Invited) Different Fluorescent Modifications of Carbon Nanotubes with Aromatic Reactants

Covalent functionalization of single-wall carbon nanotubes (SWCNTs) can produce a stable material with electronic and optical properties enhanced purposely as compare to original species. In the present report we describe a new category of reactions between photoexcited aromatic compounds and SWCNTs, creating different fluorescent trapping states in SWCNTs. The reactions take place in aqueous suspensions of SWCNTs with small addition of soluble or even very-slightly soluble organic aromatic compounds excited by UV light. The reaction rate depends on the aromatic compound structure and proportional to its concentration, and in some cases, as with aniline or iodoaniline, these strong spectral sidebands appear within one minute. Total SWCNT photoluminescence intensity can be increased by a factor as large as 5. Shifted emission bands and the trapping nature of them may be interesting for a variety of optical and electronic applications.Most notably, in most cases of these photochemical reactions, the emission spectra of altered SWCNTs become different, never observed previously, upon removal of molecular oxygen from the solution, thus indicating different chemical products of the reactions. For instance, for (6,5) SWCNTs, treatment in oxygen presence gives a new emission band red-shifted by 160 meV from the pristine position, which is similar to many previously studied reactions starting with oxygen doping. However, same UV treatments after dissolved molecular oxygen removal will lead to two new emission bands red-shifted by 140 and 270 meV, growing with different rate under UV illumination. Variance spectroscopy shows the presence of individual “multicolor” nanotubes with three distinct emission bands, original plus two shifted. The reaction demonstrates new possibilities for creation different trapping states on individual nanotubes, and opens new options for tuning electronic and optical properties of nanotubes for possible applications.

Bi- and Trifunctional Lipids for Visualization of Sphingolipid Dynamics within the Cell.

Investigations into lipid localization and transport are often hampered by a lack of methods and tools to faithfully visualize lipids in the context of living cells, since fluorescent modifications drastically change lipid properties. Here, we describe the use of bifunctional as well as trifunctional sphingosine to reveal its subcellular localization via crosslinking, fixation, and specific staining by click reaction with a fluorophore. Additionally, these probes allow investigations into lipid metabolism as revealed by thin-layer chromatography.

Design of peptoid analogue dimers and measure of their affinity for Grb2 SH3 domains.

This paper describes the design of the highest affinity ligands for Grb2 SH3 domains reported so far. These compounds were designed by combining N-alkyl amino acid incorporation in a proline-rich sequence with subsequent dimerization of the peptoid sequence based on structural data and molecular modeling. Optimization of the linker size is discussed, and the N-alkyl amino acid incorporation into both monomeric halves is reported. Because the affinity for Grb2 of the optimized compounds was too high to be measured using the fluorescent modifications that they induce on the Grb2 emission spectrum, a competition assay was developed. In this test, Grb2 is pulled down from a cellular extract by the initial VPPPVPPRRR peptide bound to Sepharose beads. In the presence of competitors, the test quantifies the amount of Grb2 displaced from the beads. It has enabled us to determine a K(i) value in the 10(-10) M range for the highest affinity Grb2 peptoid analogue dimer.

Radio-induced structural membrane modifications: a potential bioindicator of ionizing radiation exposure?

Purpose: This study focused on radio-induced membrane alterations in order to assess some related parameters as potential biological indicators of ionizing radiation effects in cases of accidental overexposure. Materials and methods: Radio-induced membrane alterations were assessed after gamma-irradiation of human blood. Biophysical techniques based on fluorescent probe incorporation into isolated living lymphocytes and erythrocytes membranes were applied. Results: Using the technique of fluorescence polarization, the lipophilic phase of the membrane was shown to be more fluid whereas the lipid-protein interface of the membrane was shown to be more rigid after gamma-irradiation. Fluorescent anisotropy modifications showed dose-time effect relationships after radiation exposure. Ionizing radiation induced a decrease in steadystate anisotropy values but did not affect the probe's lifetime as assessed by fluorescence lifetime distribution technique. These data suggest that the anisotropy variations are representative of the local properties of the fluorescent probe's micro-environment. However, the distribution width showed a decrease pointing towards radiation-induced changes of membrane domain organization, probably due not only to membrane water penetration related to lipoperoxidation, but also to compositional changes and redistribution of membrane components. In contrast, the lack of radiation effect observed using the lateral diffusion index technique may be related to the integrated overview of the radio-induced modifications of the membrane provided by this technique, which pointed out radio-induced damage to the membrane in micro-domains. Conclusion: These findings suggest the utility of structural membrane modification measurements as an early bio-indicator of ionizing radiation exposure.

Specific and fluorescent modifications of cytidine.

Specific and fluorescent modifications of cytidine.

New tests for microscale identification of anthocyanidins on thin-layer chromatograms

Five color tests, three of which are based on exposure to NH 3, one on ammonium molybdate, and the other on lead acetate for identification of the six common anthocyanidins in quantities far below 1 μg, are presented. Anthocyanidin fluorescence is intensified by an NH 3 chamber test so that concentrations well below the visible range can be detected. Each of the six anthocyanidins is identified individually by its characteristic serial fluorescent modifications produced on treatment with formic acid or sulfuric acid prior to extended NH 3 exposure. The visible and u.v. colors produced following ammonium molybdate or lead acetate sprays are distinctive, and also diagnostic of O-dihydroxylated anthocyanidins.