One Sentence Summary: Oligomers of α-synuclein generate neuronal damage when insertion of a highly structured core causes disruption of membrane integrity. This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAASAbstract: Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state NMR techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and in primary cortical neurons.

DNA hairpin-loop structures fluctuate between different conformations and are generally classified as open or closed (as shown in Fig. 1). They are involved in various biological functions, including gene expression and regulation (5, 6), and more recently they have found use as DNA biosensors (e.g., molecular beacons) (7,8).The open-to-closed transition provides a simple case with which to study the dynamics of intramolecular chain diffusion. Continuing this theme, we developed a method for detecting biopolymer conformational dynamics based on the fluctuations in proximity ratio from FRET (10). By attaching donor and acceptor fluorophores to both ends of a DNA hairpin, the open-to-closed conformational dynamics of the system can be detected at ultra-high sensitivity down to the single-molecule level. By constructing the autocorrelation function of the proximity ratio rather than of the fluorescence intensity, we simplify the extraction of intramolecular kinetics from the correlation function. The use of the ratiometric method should result in a correlation function independent of molecular diffusion (10). This new approach enables us to observe stretched exponential kinetics for the conformational fluctuation in a DNA hairpinloop system (10). A multiple-pathway, two-state model was proposed and used to simulate experimental single-molecule proximity ratio distributions (11).In our previous studies, we concentrated on the time scale of the observed fluctuation and did not fully exploit the amplitude of the autocorrelation function. Here, we report the investigation of temperature and viscosity dependence for the conformational fluctuation of a DNA hairpin-loop. We show that the correlation amplitude is directly related to the equilibrium constant of the open-to-closed transition. By using a two-state model we are able to recover the apparent thermodynamic and kinetic parameters for the hairpin-loop conformational fluctuations under different buffer conditions. The kinetics for the open-to-closed transition of the hairpin-loop appear to show non-Arrhenius behavior akin to that found in peptide ␤-hairpins (12). Materials and MethodsA 40-base oligonucleotide 5Ј-GGGTT-(A) 30 -AACCC-3Ј was chosen as our model DNA hairpin-loop. Donor fluorophore carboxytetramethylrhodamine (TMR) is attached at its 3Ј end via a modified cytosine and a six-carbon linker. Acceptor fluorophore indodicarbocyanine (Cy5) is attached at its 5Ј end via a three-carbon linker. The donor and acceptor form a fluorescence resonance energy transfer pair with a FRET distance (R 0 ) of Ϸ5.3 nm. Both dual-labeled and singly (TMR) labeled oligonucleotides were purchased from Operon Technologies (Alameda, CA) and were HPLC purified. The structure of the fully closed hairpin-loop is illustrated in Fig. 1.The viscosity was varied by adding up to 55% (in mass) of glycerol (molecular biology reagent from Sigma) to the aqueous solution. The precise viscosity of the mixture was calculated by using a polynomial fit to the tabulated viscosity of water͞glycerol m...

We have investigated the structure and unfolding kinetics of the human telomeric intramolecular G quadruplex by using singlemolecule fluorescence resonance energy transfer. An exploration of conformational heterogeneity revealed two stable folded conformations, in both sodium-and potassium-containing buffers, with small differences between their enthalpies and entropies. Both folded conformations can be opened by the addition of a 21-base complementary DNA oligonucleotide. The unfolding of both substates occurs at the same rate, which showed dependence on the monovalent metal cation present. Temperature-dependence studies in 100 mM KCl gave an apparent activation enthalpy and entropy of 6.4 ؎ 0.4 kcal⅐mol ؊1 and ؊52.3 ؎ 1.4 cal⅐mol ؊1 ⅐K ؊1 , respectively, indicating that the unfolding is entropically driven and can occur easily. In contrast, in 100 mM NaCl the respective values are 14.9 ؎ 0.2 kcal⅐mol ؊1 and ؊23.0 ؎ 0.8 cal⅐mol ؊1 ⅐K ؊1 , suggesting a more significant enthalpic barrier. Molecular modeling suggests that the two species are likely to be the parallel and antiparallel quadruplex structures. The unfolding free energy barrier is estimated to be between 3 and 15 k BT based on Kramers' theory. We conclude that under near-physiological conditions these structures coexist and can interconvert on a minute time scale. DNA sequences with stretches of multiple guanines can form four-stranded structures called quadruplexes (1). The evidence is growing that quadruplexes may be important for several biological mechanisms (1-8). Evidence supports the occurrence of quadruplexes in the cell nucleus (9), and, furthermore, a recent study suggests that particular quadruplexes may have the potential to control gene expression (10). The intramolecular DNA quadruplex based on the human telomeric repeat sequence, d(TTAGGG), has been the subject of extensive biophysical and biological studies. It has been demonstrated, in vitro, that formation of this structure impedes the extension mechanism of human telomerase (3). Furthermore, stabilization of this quadruplex may be a natural or unnatural mechanism to influence the regulation of teleomeres at the ends of chromosomes. Because telomerase function and telomere maintenance are critical for the division of cancer cells, the human telomeric intramolecular quadruplex (HTIQ) is being seriously considered as a potential molecular target for the development of novel anticancer agents (7,8). Detailed structural studies have provided evidence for two distinct folds for the HTIQ in the presence of sodium and potassium ions (11,12). An NMR spectroscopic study, where the dominant monovalent cation was sodium, showed that the HTIQ exists predominantly with an antiparallel arrangement of strands with a diagonal loop across a terminal tetrad and edgewise loops (11) (see Fig. 7A, which is published as supporting information on the PNAS web site). In contrast, a recent x-ray crystal structure of the same oligonucleotide, in the presence of potassium, shows all four strands to be parallel ...

Although polymerase chain reaction (PCR) is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications. Isothermal DNA amplification techniques are hence valuable for on-site diagnostic applications in place of traditional PCR. Here we describe a true isothermal approach for amplifying and detecting double-stranded DNA based on a CRISPR–Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification method (namely CRISDA). CRISDA takes advantage of the high sensitivity/specificity and unique conformational rearrangements of CRISPR effectors in recognizing the target DNA. In combination with a peptide nucleic acid (PNA) invasion-mediated endpoint measurement, the method exhibits attomolar sensitivity and single-nucleotide specificity in detection of various DNA targets under a complex sample background. Additionally, by integrating the technique with a Cas9-mediated target enrichment approach, CRISDA exhibits sub-attomolar sensitivity. In summary, CRISDA is a powerful isothermal tool for ultrasensitive and specific detection of nucleic acids in point-of-care diagnostics and field analyses.

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 of the two excitation lasers were carefully matched so that the volume overlap was 30% of the total confocal volume illuminated. This method greatly reduces the level of background fluorescence and, hence, extends the sensitivity of single molecule detection down to 50 fM. At these concentrations, the dual-labeled DNA is detectable in the presence of a 1000-fold excess of single-fluorophore-labeled DNA. We demonstrate that we can detect 100 fM dual-labeled DNA diluted in 1 microM unlabeled DNA, which was not possible with single color detection. This method can be used to detect rare molecules in complex mixtures.