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https://www.readbyqxmd.com/read/28931002/enhanced-proofreading-governs-crispr-cas9-targeting-accuracy
#1
Janice S Chen, Yavuz S Dagdas, Benjamin P Kleinstiver, Moira M Welch, Alexander A Sousa, Lucas B Harrington, Samuel H Sternberg, J Keith Joung, Ahmet Yildiz, Jennifer A Doudna
The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing(1-4). High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown(5-9). Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state(10) when bound to mismatched targets...
September 20, 2017: Nature
https://www.readbyqxmd.com/read/28930678/dna-ligase-iv-guides-end-processing-choice-during-nonhomologous-end-joining
#2
Michael P Conlin, Dylan A Reid, George W Small, Howard H Chang, Go Watanabe, Michael R Lieber, Dale A Ramsden, Eli Rothenberg
Nonhomologous end joining (NHEJ) must adapt to diverse end structures during repair of chromosome breaks. Here, we investigate the mechanistic basis for this flexibility. DNA ends are aligned in a paired-end complex (PEC) by Ku, XLF, XRCC4, and DNA ligase IV (LIG4); we show by single-molecule analysis how terminal mispairs lead to mobilization of ends within PECs and consequent sampling of more end-alignment configurations. This remodeling is essential for direct ligation of damaged and mispaired ends during cellular NHEJ, since remodeling and ligation of such ends both require a LIG4-specific structural motif, insert1...
September 19, 2017: Cell Reports
https://www.readbyqxmd.com/read/28930465/single-molecule-based-white-light-emissive-organic-solids-with-molecular-packing-dependent-thermally-activated-delayed-fluorescence
#3
Yuewei Zhang, Yang Miao, Xiaoxian Song, Yu Gao, Zuolun Zhang, Kaiqi Ye, Yue Wang
White-light emitting single molecules have attracted broad attention because of their great potential for use in flat-panel displays and future light sources. Herein, we report a unique molecule of 3-(diphenylamino)-9H-xanthen-9-one (3-DPH-XO), which was found to exhibit bright white-light emission in the solid state caused by the spontaneous formation of a mixture with different polymorphs. Single-crystal analyses demonstrate that non-covalent interactions (such as π•••π stacking, hydrogen bonding, and C-H•••π interactions) induce different stacking arrangements (polymorphs A, B, and C) with different photophysical properties in a molecular solid...
September 20, 2017: Journal of Physical Chemistry Letters
https://www.readbyqxmd.com/read/28930438/supported-planar-mammalian-membranes-as-models-of-in-vivo-cell-surface-architectures
#4
Han-Yuan Liu, Hannah Grant, Hung-Lun Hsu, Raya Sorkin, Filip Bošković, G J L Wuite, Susan Daniel
Emerging technologies use cell plasma membrane vesicles or 'blebs' as an intermediate to form molecularly complete, planar cell surface mimetics that are compatible with a variety of characterization tools and microscopy methods. This approach enables direct incorporation of membrane proteins into supported lipid bilayers without using detergents and reconstitution and preserves native lipids and membrane species. Such a system can be advantageous as in vitro models of in vivo cell surfaces for study of the roles of membrane proteins as drug targets in drug delivery, host-pathogen interactions, tissue engineering, and many other bioanalytical and sensing applications...
September 20, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28928405/nanopore-extended-field-effect-transistor-for-selective-single-molecule-biosensing
#5
Ren Ren, Yanjun Zhang, Binoy Paulose Nadappuram, Bernice Akpinar, David Klenerman, Aleksandar P Ivanov, Joshua B Edel, Yuri Korchev
There has been a significant drive to deliver nanotechnological solutions to biosensing, yet there remains an unmet need in the development of biosensors that are affordable, integrated, fast, capable of multiplexed detection, and offer high selectivity for trace analyte detection in biological fluids. Herein, some of these challenges are addressed by designing a new class of nanoscale sensors dubbed nanopore extended field-effect transistor (nexFET) that combine the advantages of nanopore single-molecule sensing, field-effect transistors, and recognition chemistry...
September 19, 2017: Nature Communications
https://www.readbyqxmd.com/read/28927531/discriminating-rna-variants-with-single-molecule-allele-specific-fish
#6
REVIEW
Martyna O Urbanek, Wlodzimierz J Krzyzosiak
DNA mutations of various types often affect the cellular localization and function of gene products. The role of mutant transcripts in the pathogenesis of human disease is increasingly recognized. Among the pathogenic RNA variants are transcripts with single nucleotide substitutions, small insertions or deletions, aberrantly or alternatively spliced transcripts and RNAs derived from fused genes. To discriminate among transcripts, particularly those of low abundance, showing small or large sequence differences, a highly sensitive and specific RNA imaging method is required...
July 2017: Mutation Research
https://www.readbyqxmd.com/read/28927527/dna-mismatch-repair-and-its-many-roles-in-eukaryotic-cells
#7
REVIEW
Dekang Liu, Guido Keijzers, Lene Juel Rasmussen
DNA mismatch repair (MMR) is an important DNA repair pathway that plays critical roles in DNA replication fidelity, mutation avoidance and genome stability, all of which contribute significantly to the viability of cells and organisms. MMR is widely-used as a diagnostic biomarker for human cancers in the clinic, and as a biomarker of cancer susceptibility in animal model systems. Prokaryotic MMR is well-characterized at the molecular and mechanistic level; however, MMR is considerably more complex in eukaryotic cells than in prokaryotic cells, and in recent years, it has become evident that MMR plays novel roles in eukaryotic cells, several of which are not yet well-defined or understood...
July 2017: Mutation Research
https://www.readbyqxmd.com/read/28926278/planar-optical-nano-antennas-resolve-cholesterol-dependent-nanoscale-heterogeneities-in-the-plasma-membrane-of-living-cells
#8
Raju Regmi, Pamina Martina Winkler, Valentin Flauraud, Kyra Borgman, Carlo Manzo, Juergen Brugger, Herve Rigneault, Jerome Wenger, Maria F Garcia-Parajo
Optical nano-antennas can efficiently confine light into nanoscopic hotspots enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nano-antenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale...
September 19, 2017: Nano Letters
https://www.readbyqxmd.com/read/28926050/temperature-dependence-of-the-conversion-efficiency-of-photochromic-perylene-bisimide-dithienylcyclopentene-triads-embedded-in-a-polymer
#9
Johann Thurn, Johannes Maier, Martti Pärs, Katja Gräf, Mukundan Thelakkat, Jürgen Köhler
Photochromic molecules that are covalently linked to a strong fluorophore combine the requirements of external control and strong fluorescence, which will become increasingly important for super-resolution microscopy techniques based on single molecules. However, given the bulky structure of such constructs, steric hindrance might affect their photoconversion efficiencies upon immobilising them for imaging purposes. In this study the efficiencies of the photochromic conversion processes of molecular triads that are embedded in a polymer have been studied as a function of temperature...
September 19, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/28924672/in-situ-super-resolution-imaging-of-genomic-dna-with-oligostorm-and-oligodna-paint
#10
Brian J Beliveau, Alistair N Boettiger, Guy Nir, Bogdan Bintu, Peng Yin, Xiaowei Zhuang, C-Ting Wu
OligoSTORM and OligoDNA-PAINT meld the Oligopaint technology for fluorescent in situ hybridization (FISH) with, respectively, Stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) to enable in situ single-molecule super-resolution imaging of nucleic acids. Both strategies enable ≤20 nm resolution and are appropriate for imaging nanoscale features of the genomes of a wide range of species, including human, mouse, and fruit fly (Drosophila)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924670/correlative-single-molecule-localization-microscopy-and-confocal-microscopy
#11
Christian Soeller, Yufeng Hou, Isuru D Jayasinghe, David Baddeley, David Crossman
Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924669/measuring-nanometer-distances-between-fluorescent-labels-step-by-step
#12
Susanna Maria Früh, Ingmar Schoen
Super-resolution fluorescence microscopy methods are increasingly applied to study the structure of biological molecules within their natural context or at biomaterial interfaces. We here provide a protocol for Single-molecule High-Resolution Imaging with Photobleaching (SHRImP) that can be used to obtain information about the conformation of large proteins or other macromolecules at the single-molecule level. This procedure requires site-specific protein labeling with fluorescent dyes, immobilization and sample preparation, optimization of imaging buffer composition and microscope settings, and acquisition of short time-lapse movies that capture the stepwise bleaching behavior of individual molecules...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924668/synthesis-of-janelia-fluor-halotag-and-snap-tag-ligands-and-their-use-in-cellular-imaging-experiments
#13
Jonathan B Grimm, Timothy A Brown, Brian P English, Timothée Lionnet, Luke D Lavis
The development of genetically encoded self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in microscopy. Intracellular labeling using these systems requires small, cell-permeable dyes with high brightness and photostability. We recently discovered a general method to improve the properties of classic fluorophores by replacing N,N-dimethylamino groups with four-membered azetidine rings to create the "Janelia Fluor" dyes. Here, we describe the synthesis of the HaloTag and SNAP-tag ligands of Janelia Fluor 549 and Janelia Fluor 646 as well as standard labeling protocols for use in ensemble and single-molecule cellular imaging...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924667/correlative-in-resin-super-resolution-fluorescence-and-electron-microscopy-of-cultured-cells
#14
Errin Johnson, Rainer Kaufmann
Correlative super-resolution light and electron microscopy (super-resolution CLEM) is a powerful and emerging tool in biological research. The practical realization of these two very different microscopy techniques with their individual requirements remains a challenging task. There is a broad range of approaches to choose from, each with their own advantages and limitations. Here, we present a detailed protocol for in-resin super-resolution CLEM of high-pressure frozen and freeze substituted cultured cells...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924666/brain-slice-staining-and-preparation-for-three-dimensional-super-resolution-microscopy
#15
Christopher L German, Manasa V Gudheti, Annette E Fleckenstein, Erik M Jorgensen
Localization microscopy techniques-such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM)-provide the highest precision for single-molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924665/fully-automated-targeted-confocal-and-single-molecule-localization-microscopy
#16
Jan Philipp Eberle, Walter Muranyi, Holger Erfle, Manuel Gunkel
Single-molecule localization microscopy (SMLM) enables imaging of biological structures in the nanometre range. Long measurement times are the consequence of this kind of microscopy due to the need of acquiring thousands of images. We built a setup that automatically detects target structures using confocal microscopy and images them with SMLM. Utilizing the Konstanz Information Miner (KNIME), we were able to connect a confocal microscope with an SMLM unit for targeted screening. In this process, we developed KNIME plugins to communicate with the microscope components and combined them to a workflow...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924664/two-color-single-molecule-tracking-in-live-cells
#17
Siegfried Hänselmann, Dirk-Peter Herten
Measuring the kinetics of protein-protein interactions between molecules in the plasma membrane of live cells provides valuable information for understanding dynamic processes, like cellular signaling, on a molecular scale. Two-color single-molecule tracking is a fluorescence microscopy-based method to detect and quantify specific protein-protein interactions on a single-event level, providing sensitivity to heterogeneities and rare events. Fundamentally, it allows following the movement of single molecules of two different protein species in live cells with a localization precision beyond the diffraction limit of light in real time...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924663/quantitative-single-molecule-localization-microscopy-qsmlm-of-membrane-proteins-based-on-kinetic-analysis-of-fluorophore-blinking-cycles
#18
Franziska Fricke, Joel Beaudouin, Sebastian Malkusch, Roland Eils, Mike Heilemann
Photoswitchable or photoactivatable fluorophores are the key in single-molecule localization microscopy. Next to providing fluorescence images with subdiffraction spatial resolution, additional information is available from observing single fluorophores over time. This includes the characteristic photophysical phenomenon of "blinking" that is exhibited by single fluorescent proteins or fluorophores and follows well-defined kinetic laws. Analyzing the kinetics of "blinking" allows determining the number of fluorophores in a multi-molecular complex...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28924226/electrically-driven-single-photon-emission-from-an-isolated-single-molecule
#19
Li Zhang, Yun-Jie Yu, Liu-Guo Chen, Yang Luo, Ben Yang, Fan-Fang Kong, Gong Chen, Yang Zhang, Qiang Zhang, Yi Luo, Jin-Long Yang, Zhen-Chao Dong, J G Hou
Electrically driven molecular light emitters are considered to be one of the promising candidates as single-photon sources. However, it is yet to be demonstrated that electrically driven single-photon emission can indeed be generated from an isolated single molecule notwithstanding fluorescence quenching and technical challenges. Here, we report such electrically driven single-photon emission from a well-defined single molecule located inside a precisely controlled nanocavity in a scanning tunneling microscope...
September 18, 2017: Nature Communications
https://www.readbyqxmd.com/read/28924219/mesoscopic-model-for-dna-g-quadruplex-unfolding
#20
A E Bergues-Pupo, I Gutiérrez, J R Arias-Gonzalez, F Falo, A Fiasconaro
Genomes contain rare guanine-rich sequences capable of assembling into four-stranded helical structures, termed G-quadruplexes, with potential roles in gene regulation and chromosome stability. Their mechanical unfolding has only been reported to date by all-atom simulations, which cannot dissect the major physical interactions responsible for their cohesion. Here, we propose a mesoscopic model to describe both the mechanical and thermal stability of DNA G-quadruplexes, where each nucleotide of the structure, as well as each central cation located at the inner channel, is mapped onto a single bead...
September 18, 2017: Scientific Reports
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