A structural look at α-synuclein oligomers Fibrillar aggregates of the protein (αS) are major constituents Lewy bodies in Parkinson's disease. However, small that accumulate during process fibril formation thought to cause neuronal toxicity associated with onset and progression Little is known about detailed properties αS molecular mechanisms lead their toxicity. Fusco et al. report characterization two forms oligomers, which elucidates fundamental elements giving rise Science , this issue p. 1440

Abstract Although polymerase chain reaction (PCR) is the most widely used method for DNA amplification, requirement of thermocycling limits its non-laboratory applications. Isothermal amplification techniques are hence valuable on-site diagnostic applications in place traditional PCR. Here we describe a true isothermal approach amplifying and detecting double-stranded based on CRISPR–Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification (namely CRISDA). CRISDA takes...

Abstract A biodegradable two‐dimensional (2D) delivery platform based on loading black phosphorus nanosheets (BPs) with Cas9 ribonucleoprotein engineered three nuclear localization signals (NLSs) at C terminus (Cas9N3) is successfully established. The Cas9N3‐BPs enter cells effectively via membrane penetration and endocytosis pathways, followed by a BPs biodegradation‐associated endosomal escape cytosolic releases of the loaded Cas9N3 complexes. thus provide efficient genome editing gene...

We have investigated the structure and unfolding kinetics of human telomeric intramolecular G quadruplex by using single-molecule fluorescence resonance energy transfer. An exploration conformational heterogeneity revealed two stable folded conformations, in both sodium- potassium-containing buffers, with small differences between their enthalpies entropies. Both conformations can be opened addition a 21-base complementary DNA oligonucleotide. The substates occurs at same rate, which showed...

Abstract α-Synuclein (αS) is a presynaptic disordered protein whose aberrant aggregation associated with Parkinson’s disease. The functional role of αS still debated, although it has been involved in the regulation neurotransmitter release via interaction synaptic vesicles (SVs). We report here detailed characterisation conformational properties bound to inner and outer leaflets plasma membrane (PM), using small unilamellar vesicles. Our results suggest that preferentially binds PM leaflet....

Copper is an essential trace element in living organisms with its levels and localisation being carefully managed by the cellular machinery. However, if misregulated, deficiency or excess of copper ions can lead to several diseases. Therefore, it important have reliable methods detect, monitor visualise this metal cells. Herein we report a new optical probe based on BODIPY, which shows switch-on fluorescence intensity upon binding copper(I), but not presence high concentration other...

We present a new, general method for the controlled deposition of biological molecules on surfaces, based nanopipet operating in ionic solution. The potential applied to pipet tip controls flux from pipet, allowing fine control delivery rate. used ion current distance surface glass slide and deposited fluorescently labeled DNA or protein G at defined location onto surface. Features 830 nm size were obtained by depositing biotinylated streptavidin surface; 1.3 μm spots positively charged

Intramolecular chain diffusion is an elementary process in the conformational fluctuations of DNA hairpin-loop. We have studied temperature and viscosity dependence a model hairpin-loop by FRET (fluorescence resonance energy transfer) fluctuation spectroscopy (FRETfs). Apparent thermodynamic parameters were obtained analyzing correlation amplitude through two-state are consistent with steady-state fluorescence measurements. The kinetics closing loop show non-Arrhenius behavior, agreement...

We have detected individual DNA molecules labeled with two different fluorophores in solution by using two-color excitation and detection of coincidence fluorescence bursts. The confocal volumes the lasers were carefully matched so that volume overlap was 30% total illuminated. This method greatly reduces level background and, hence, extends sensitivity single molecule down to 50 fM. At these concentrations, dual-labeled is detectable presence a 1000-fold excess single-fluorophore-labeled...

We show that direct coupling of a dye-labelled DNA (acceptor) to quantum dot (QD) donor significantly reduces the donor–acceptor distance and improves FRET efficiency: highly efficient (∼88%) at low acceptor-to-donor ratio 2 has been achieved single-molecule level.

We report a study of the allophycocyanin trimer (APC), light-harvesting protein complex from cyanobacteria, by room-temperature single-molecule measurements fluorescence spectra, lifetimes, intensity trajectories, and polarization modulation. Emission spectra individual APC trimers are found to be homogeneous on time scale seconds. In contrast, their emission lifetimes widely distributed because generation long-lived exciton traps during course measurements. The trajectories modulation...

In this letter, we report the preparation of a compact, functional quantum dot (QD)−DNA conjugate, where capturing target DNA is directly and covalently coupled to QD surface. This enables control separation distance between donor dye acceptor within range Förster radius. Moreover, tri(ethylene glycol) linker introduced surface coating effectively eliminate strong, nonspecific adsorption on As result, QD−DNA conjugate hybridizes specifically its complementary with hybridization rate constant...

Herein we report on the single-molecule analysis of a conserved DNA quadruplex element found in transcriptional activation site promoter proto- oncogene c-kit. We demonstrate that c-kit motif is able to fold into nonduplex states within natural extended duplex. There are apparent differences putative structures, and K+ dependence formation, appear result when studying this duplex, as opposed single-stranded form.

Caught in a trap and on camera: A nanopipette is used for electrodeless dielectrophoresis clear evidence shown, by using wide-field fluorescence imaging (see image), the reversible trapping of Alexa-488-labeled proteins (protein G immunoglobulin G) also fluorophore alone. The dielectrophoretic concentration enhanced at least factor 300 these fluorophore-labeled proteins. Supporting Information this article available WWW under http://www.wiley-vch.de/contents/jc_2002/2005/z500196_s.html or...

The tumor suppressor p53 is a member of the emerging class proteins that have both folded and intrinsically disordered domains, which are challenge to structural biology. Its N-terminal domain (NTD) linked core domain, has link tetramerization followed by C-terminal domain. quaternary structure human been solved combination NMR spectroscopy, electron microscopy, small-angle X-ray scattering (SAXS), NTD ensemble SAXS. murine reported different structure, with N C termini interacting. Here, we...

We have used a nanopipet as nanopen to locally and controllably deposit complex biomolecules, including antibodies DNA, onto surface create multicomponent functional submicron features. Key advantages of this method are that the biomolecules always in solution applied voltage provides fine control delivery down single molecule level. Two consecutive cycles deposition, produce spatially varying features with different biological properties, were demonstrated fluorescently labeled or biotin....

The boundary between the physical and biological sciences has been eroded in recent years with new methods applied to biology molecules being used for purposes. We have pioneered application of a form scanning probe microscopy based on scanned nanopipette, originally developed by Hansma co-workers, reliable non-contact imaging over surface live cell. found that nanopipette can also be controlled local voltage-driven reagents or biomolecules this deposition delivery probes mapping specific...

The kinetics of opening the DNA quadruplex formed by human telomeric repeat have been investigated using real-time fluorescence resonance energy transfer (FRET) measurements with a peptide nucleic acid (PNA) trap. It has found that this is zero-order respect to PNA, indicating initial step rate-limiting internal rearrangement quadruplex. A study temperature dependence rate was performed and activation process estimated be 98 ± 8 kJ mol-1.

Conventional cooperative protein folding invokes discrete ensembles of native and denatured state structures in separate free-energy wells. Unimodal noncooperative ("downhill") folding, however, proposes an ensemble states occupying a single well for proteins at >/=4 x 10(4) s(-1) 298 K. It is difficult to falsify unimodal mechanisms such fast by standard equilibrium experiments because both can present the same time-averaged structural, spectroscopic, thermodynamic properties when time...

We report the measurement of surface conductivity biological macromolecules by dielectrophoretic trapping at tip a glass nanopipet. find that threshold voltage for is function salt concentration and can be directly linked to effective biomolecule its solvation shell. The conductivities obtained 20-mer single-stranded DNA, 40-mer double-stranded yellow fluorescent protein are $7.9\ifmmode\pm\else\textpm\fi{}1.9\text{ }\text{ }\mathrm{nS}$, $5.3\ifmmode\pm\else\textpm\fi{}0.7\text{...

Abstract A biodegradable two‐dimensional (2D) delivery platform based on loading black phosphorus nanosheets (BPs) with Cas9 ribonucleoprotein engineered three nuclear localization signals (NLSs) at C terminus (Cas9N3) is successfully established. The Cas9N3‐BPs enter cells effectively via membrane penetration and endocytosis pathways, followed by a BPs biodegradation‐associated endosomal escape cytosolic releases of the loaded Cas9N3 complexes. thus provide efficient genome editing gene...

Protein aggregation is a hallmark of major neurodegenerative disorders. Increasing data suggest that smaller aggregates cause higher toxic response than filamentous (fibrils). However, the size small has challenged their detection within biologically relevant environments. Here, we report approaches to quantitatively super-resolve in live cells and ex vivo brain tissues. We show Amytracker 630 (AT630), commercial aggregate-activated fluorophore, outstanding photophysical properties enable...

α-Synuclein (α-Syn) is an intrinsically disordered protein whose aggregation in the brain has been significantly implicated Parkinson's disease (PD). Beyond brain, oligomers of are also found cerebrospinal fluid (CSF) and blood, where analysis these aggregates may provide diagnostic routes enable a better understanding mechanisms. However, detecting α-Syn CSF blood challenging due to its heterogeneous size shape, low abundance clinical samples. Nanopore technology offers promising route for...

The motions of a dye-labeled DNA hairpin loop (Cy5-5'-GGGTT-(A)30-AACCC-3'-TMR) have been investigated through the fluctuations in proximity ratio from fluorescence resonance energy transfer (FRET). We examine three solution conditions: (1) MilliQ water, (2) Tris-EDTA buffer, and (3) buffer plus an excess complementary to sequence, (T)30. Correlations show submillisecond dynamics. Static heterogeneity is revealed distribution amplitudes. observed stretched exponential kinetics are consistent...