Cholesterol and sphingomyelin form together a highly ordered membrane phase, which is believed to play important biological functions in plasma membranes of mammalian cells. Since sphingomyelin is present mainly at the outer leaflet of cell membranes, monitoring its lipid order requires molecular probes capable to bind specifically at this leaflet and exhibit negligibly slow flip-flop. In the present work, such a probe was developed by modifying the solvatochromic fluorescent dye Nile Red with an amphiphilic anchor group. To evaluate the flip-flop of the obtained probe (NR12S), we developed a methodology of reversible redox switching of its fluorescence at one leaflet using sodium dithionite. This method shows that NR12S, in contrast to parent Nile Red, binds exclusively the outer membrane leaflet of model lipid vesicles and living cells with negligible flip-flop in the time scale of hours. Moreover, the emission maximum of NR12S in model vesicles exhibits a significant blue shift in liquid ordered phase (sphingomyelin-cholesterol) as compared to liquid disordered phase (unsaturated phospholipids). As a consequence, these two phases could be clearly distinguished in NR12S-stained giant vesicles by fluorescence microscopy imaging of intensity ratio between the blue and red parts of the probe emission spectrum. Being added to living cells, NR12S binds predominantly, if not exclusively, their plasma membranes and shows an emission spectrum intermediate between those in liquid ordered and disordered phases of model membranes. Importantly, the emission color of NR12S correlates well with the cholesterol content in cell membranes, which allows monitoring the cholesterol depletion process with methyl-beta-cyclodextrin by fluorescence spectroscopy and microscopy. The attractive photophysical and switching properties of NR12S, together with its selective outer leaflet staining and sensitivity to cholesterol and lipid order, make it a new powerful tool for studying model and cell membranes.
The current challenge in the field of fluorescent nanoparticles (NPs) for bioimaging is to achieve extreme brightness and external control of their emission using biodegradable materials. Here we propose a new concept of fluorescent polymer NPs, doped with ionic liquid-like salts of a cationic dye (octadecyl rhodamine B) with a bulky hydrophobic counterion (fluorinated tetraphenylborate) that serves as spacer minimizing dye aggregation and self-quenching. The obtained 40-nm poly(D,L-lactide-co-glycolide) NPs containing up to 500 dyes are brighter than quantum dots and exhibit photo-induced reversible on/off fluorescence switching, never reported for dye-doped NPs. We show that this collective switching of hundreds of dyes is due to ultrafast excitation energy transfer and can be used for super-resolution imaging. These NPs, being spontaneously endocytosed by living cells, feature high signal-to-noise ratio and absence of toxicity. The counterion-based concept opens the way to a new class of nanomaterials for sensing, imaging and light harvesting.
We review the main trends in the development of fluorescence probes to obtain information about the structure, dynamics, and interactions in biomembranes. These probes are efficient for studying the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location. Progress is being made in increasing the information content and spatial resolution of the probe responses. Multichannel environment-sensitive probes that can distinguish between different membrane physicochemical properties through multiple spectroscopic parameters show considerable promise.
Polyethylenimines (PEI) constitute efficient nonviral vectors for gene transfer. However, because free PEI shows some cytotoxicity and because intracellular dissociation of PEI/DNA complexes seems to be required for efficient transfection, it is important to monitor the concentrations of free and bound partners in the mixtures of DNA and PEI used for transfection. To reach this objective, we used fluorescence correlation spectroscopy with two-photon excitation to characterize the complexes formed with either rhodamine-labeled 25 kDa PEI or DNA plasmid molecules. At the molar ratios of PEI nitrogen atoms to DNA phosphate usually used for transfection, we found that approximately 86% of the PEI molecules were in a free form. The PEI/DNA complexes are composed on the average by 3.5 (+/-1) DNA plasmids and approximately 30 PEI molecules. From this composition and the pK(a) of PEI, it could be inferred that in contrast to DNA condensation by small multivalent cations, only a limited neutralization of the DNA phosphate groups is required for DNA condensation by PEI. Moreover, DNA appears only poorly compacted in the PEI/DNA complexes. As an application, fluorescence correlation spectroscopy was used to monitor the purification of PEI/DNA complexes by ultrafiltration as well as the heparin-induced dissociation of the complexes.
Herein, we developed the first ratiometric fluorescent probe for apoptosis detection. This probe incorporates selectively into the outer leaflet of the cell plasma membrane and senses the loss of the plasma membrane asymmetry occurring during the early steps of apoptosis. The high specificity to the plasma membranes was achieved by introduction into the probe of a membrane anchor, composed of a zwitterionic group and a long (dodecyl) hydrophobic tail. The fluorescence reporter of this probe is 4'-(diethylamino)-3-hydroxyflavone, which exhibits excited-state intramolecular proton transfer (ESIPT), resulting in two-band emission highly sensitive to the lipid composition of the biomembranes. Fluorescence spectroscopy, flow cytometry, and microscopy measurements show that the ratio of the two emission bands of the probe changes dramatically in response to apoptosis. This response reflects the changes in the lipid composition of the outer leaflet of the cell plasma membrane because of the exposure of the anionic phospholipids from the inner leaflet at the early steps of apoptosis. Being ratiometric, the response of the new probe can be easily quantified on an absolute scale. This allows monitoring by laser scanning confocal microscopy the degree and spatial distribution of the apoptotic changes at the cell plasma membranes, a feature that can be hardly achieved with the commonly used fluorescently labeled annexin V assay.
Here, we explore the enhancement of single molecule emission by polymeric nano-antenna that can harvest energy from thousands of donor dyes to a single acceptor. In this nano-antenna, the cationic dyes are brought together in very close proximity using bulky counterions, thus enabling ultrafast diffusion of excitation energy (≤30 fs) with minimal losses. Our 60-nm nanoparticles containing >10,000 rhodamine-based donor dyes can efficiently transfer energy to 1-2 acceptors resulting in an antenna effect of ~1,000. Therefore, single Cy5-based acceptors become 25-fold brighter than quantum dots QD655. This unprecedented amplification of the acceptor dye emission enables observation of single molecules at illumination powers (1-10 mW cm-2) that are >10,000-fold lower than typically required in single-molecule measurements. Finally, using a basic setup, which includes a 20X air objective and a sCMOS camera, we could detect single Cy5 molecules by simply shining divergent light on the sample at powers equivalent to sunlight.
The critical functions of the HIV-1 nucleocapsid protein NCp7 in genomic RNA packaging and reverse transcription, essentially rely on interactions with nucleic acids. A significant progress in the knowledge of these interactions has been recently achieved with the NMR-derived structures of NCp7 derivatives in complex with two short sequences of the HIV-1 psi packaging signal, namely ACGCC and the stem-loop 3 (SL3) motif. To further identify the key nucleotides in the formation of both NCp7-d(ACGCC) and NCp7-SL3 complexes, we quantitatively analyzed by steady-state and time-resolved fluorescence, the interaction of NCp7 with d(ACGCC) and SL3 mutants where each nucleotide in interaction with the protein has been systematically substituted. Moreover, by using several NCp7 derivatives, we investigated the contributions of Phe16, Trp37, and Trp61, and the various NCp7 domains, in the binding process. The binding of NCp7 appeared essentially driven by the interaction of the zinc finger domain and notably Trp37 with a G residue, irrespective of its location in the oligonucleotide. The involvement of Trp37 in the binding process depended on its location in the C-terminal finger motif and the proper folding of this motif. Phe16 in the N-terminal finger motif also strongly contributed to the binding energy, while in contrast, Trp61 in the C-terminal domain only marginally interacted with the oligonucleotides. The stem-loop structure of SL3 stabilized the binding of NCp7 by about -7 kJ/mol (at 0.1 M NaCl) by favoring the electrostatic binding of both N- and C-terminal domains. Finally, we found that NCp7 bound to nucleic acid single-stranded regions with the following preference: X(i)()TGX(j)() > X(i)()GXGX(j)() approximately X(i)()TXGX(j)() > X(i)()GX(j)() >> X(i)()X(j)(), where X corresponds to either A or C. This implies that recognition of nucleic acids by NCp7 may be achieved by a limited number of sites, and hence, no strong affinities are required in order to get a selective binding.
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