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Current Protocols in Chemical Biology

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https://www.readbyqxmd.com/read/29241296/evolving-aptamers-with-unnatural-base-pairs
#1
Michiko Kimoto, Ken-Ichiro Matsunaga, Ichiro Hirao
A novel technology, genetic alphabet expansion, has rapidly advanced through the successful creation of unnatural base pairs that function as a third base pair in replication. Recently, genetic alphabet expansion has been applied to some practical areas. Among them, the application to DNA aptamer generation is a good example of the broad utility of this technology. A hydrophobic unnatural base pair, Ds-Px, which exhibits high fidelity in replication as a third base pair, has been applied to an evolutionary engineering method called SELEX (Systematic Evolution of Ligands by EXponential enrichment) to generate DNA aptamers that bind to targets...
December 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/29241295/in-vitro-selection-and-characterization-of-dna-aptamers-to-a-small-molecule-target
#2
Annamaria Ruscito, Erin M McConnell, Anna Koudrina, Ranganathan Velu, Christopher Mattice, Vernon Hunt, Maureen McKeague, Maria C DeRosa
Aptamers, synthetic oligonucleotide-based molecular recognition probes, have found use in a wide array of biosensing technologies based on their tight and highly selective binding to a variety of molecular targets. However, the inherent challenges associated with the selection and characterization of aptamers for small molecule targets have resulted in their underrepresentation, despite the need for small molecule detection in fields such as medicine, the environment, and agriculture. This protocol describes the steps in the selection, sequencing, affinity characterization, and truncation of DNA aptamers that are specific for small molecule targets...
December 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/29241294/early-stage-formulation-considerations
#3
Robert W Lee, Mark Mitchnick
When a drug candidate-i.e., a new chemical entity (NCE) or new molecular entity (NME)-is discovered, there is a requirement to identify a vehicle for in vitro and/or in vivo evaluation to assess the activity and/or toxicity of the compound (here we refer to the biologically active compound as the active pharmaceutical ingredient: API). Ideally, this vehicle will not impart any biological activity or any toxicity that would mask or confound the effects of the API. At this early stage in development, and given the high attrition rates of drug candidates in discovery, it does not make sense to fully characterize the API-speed and cost are generally the driving factors...
December 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/29241293/in-vivo-delivery-of-nanoparticles-into-plant-leaves
#4
Honghong Wu, Israel Santana, Joshua Dansie, Juan P Giraldo
Plant nanobiotechnology is an interdisciplinary field at the interface of nanotechnology and plant biology that aims to utilize nanomaterials as tools to study, augment or impart novel plant functions. The delivery of nanoparticles to plants in vivo is a key initial step to investigate plant nanoparticle interactions and the impact of nanoparticles on plant function. Quantum dots are smaller than plant cell wall pores, have versatile surface chemistry, bright fluorescence and do not photobleach, making them ideal for the study of nanoparticle uptake, transport, and distribution in plants by widely available confocal microscopy tools...
December 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/29241292/the-in-situ-enzymatic-screening-ises-approach-to-reaction-discovery-and-catalyst-identification
#5
Robert A Swyka, David B Berkowitz
The importance of discovering new chemical transformations and/or optimizing catalytic combinations has led to a flurry of activity in reaction screening. The in situ enzymatic screening (ISES) approach described here utilizes biological tools (enzymes/cofactors) to advance chemistry. The protocol interfaces an organic reaction layer with an adjacent aqueous layer containing reporting enzymes that act upon the organic reaction product, giving rise to a spectroscopic signal. ISES allows the experimentalist to rapidly glean information on the relative rates of a set of parallel organic/organometallic reactions under investigation, without the need to quench the reactions or draw aliquots...
December 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28910858/overview-of-methods-and-strategies-for-conducting-virtual-small-molecule-screening
#6
REVIEW
Xavier Fradera, Kerim Babaoglu
Virtual screening (VS) in the context of drug discovery is the use of computational methods to discover novel ligands with a desired biological activity from within a larger collection of molecules. These techniques have been in use for many years, there is a wide range of methodologies available, and many successful applications have been reported in the literature. VS is often used as an alternative or a complement to High-throughput screening (HTS) or other methods to identify ligands for target validation or medicinal chemistry projects...
September 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28910857/a-guide-to-quantitative-biomarker-assay-development-using-whispering-gallery-mode-biosensors
#7
Heather M Robison, Ryan C Bailey
Whispering gallery mode (WGM) sensors are a class of powerful analytical techniques defined by the measurement of changes in the local refractive index at or near the sensor surface. When functionalized with target-specific capture agents, analyte binding can be measured with very low limits of detection. There are many geometric manifestations of WGM sensors, with chip-integrated silicon photonic devices first commercialized because of the robust, wafer-scale device fabrication, facile optical interrogation, and amenability to the creation of multiplexed sensor arrays...
September 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28910856/high-throughput-screening-of-hect-e3-ubiquitin-ligases-using-ubfluor
#8
Peter K Foote, David T Krist, Alexander V Statsyuk
HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin thioesters and a complex mixture of polyubiquitin products provide further opportunities for off-target inhibition and increase the complexity of the assay. UbFluor is a novel ubiquitin thioester that bypasses the E1 and E2 enzymes and undergoes direct transthiolation with HECT E3 ligases...
September 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28910855/optimized-pei-based-transfection-method-for-transient-transfection-and-lentiviral-production
#9
Shaozhe Yang, Xiaoling Zhou, Rongxiang Li, Xiuhong Fu, Pingnan Sun
Polyethyleneimine (PEI), a cationic polymer vehicle, forms a complex with DNA which then can carry anionic nucleic acids into eukaryotic cells. PEI-based transfection is widely used for transient transfection of plasmid DNA. The efficiency of PEI-based transfection is affected by numerous factors, including the way the PEI/DNA complex is prepared, the ratio of PEI to DNA, the concentration of DNA, the storage conditions of PEI solutions, and more. Considering the major influencing factors, PEI-based transfection has been optimized to improve its efficiency, reproducibility, and consistency...
September 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28910854/characterizing-the-nano-bio-interface-using-microscopic-techniques-imaging-the-cell-system-is-just-as-important-as-imaging-the-nanoparticle-system
#10
Christie M Sayes, Henry Lujan
The rapid growth of nanotechnology and its industries has elevated the need to understand the risks associated with handling, using, and disposing of nanomaterials. These risks can be assessed through exposure measurement and hazard identification. One of the common challenges associated with quantifying nanomaterials in products, waste, humans, or the environment is the lack of tools available to measure concentration. The ability of refined tools and techniques to qualitatively detect nanoparticles in complex matrices has been demonstrated...
September 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628203/photoactivated-in-vivo-proximity-labeling
#11
David B Beck, Roberto Bonasio
Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co-purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low-affinity interactions and cannot be applied to proteins that localize to difficult-to-solubilize cellular compartments. In vivo proximity labeling (IPL) overcomes these obstacles by covalently tagging proteins and RNAs based on their proximity in vivo to a protein of interest...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628202/paper-based-invasion-assays-for-quantifying-cellular-movement-in-three-dimensional-tissue-like-structures
#12
C Chad Lloyd, Matthew W Boyce, Matthew R Lockett
To elucidate the chemical and environmental conditions that promote invasion of cancer cells, an assay is needed in which the chemical landscape of a tumor-like environment can be experimentally manipulated and probed. The three-dimensional paper-based invasion assays described here simulate poorly vascularized tissue and allow the invasion of cancerous cells to be visualized and quantified. These cultures are easy to assemble and allow multiple invasion assays to be performed in parallel. By using different materials to control gradients formed across the culture, the chemotactic potential of small molecules can be evaluated in a more representative tissue microenvironment...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628201/designing-drug-response-experiments-and-quantifying-their-results
#13
Marc Hafner, Mario Niepel, Kartik Subramanian, Peter K Sorger
We developed a Python package to help in performing drug-response experiments at medium and high throughput and evaluating sensitivity metrics from the resulting data. In this article, we describe the steps involved in (1) generating files necessary for treating cells with the HP D300 drug dispenser, by pin transfer or by manual pipetting; (2) merging the data generated by high-throughput slide scanners, such as the Perkin Elmer Operetta, with treatment annotations; and (3) analyzing the results to obtain data normalized to untreated controls and sensitivity metrics such as IC50 or GR50 ...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628200/local-generation-and-imaging-of-hydrogen-peroxide-in-living-cells
#14
Yulia A Bogdanova, Carsten Schultz, Vsevolod V Belousov
Described here is a localized H2 O2 generation-detection system consisting of a yeast D-amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal production and examination of the intracellular H2 O2 dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H2 O2 generated by the activated DAAO...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628199/measuring-cancer-drug-sensitivity-and-resistance-in-cultured-cells
#15
Mario Niepel, Marc Hafner, Mirra Chung, Peter K Sorger
Measuring the potencies of small-molecule drugs in cell lines is a critical aspect of preclinical pharmacology. Such experiments are also prototypical of high-throughput experiments in multi-well plates. The procedure is simple in principle, but many unrecognized factors can affect the results, potentially making data unreliable. The procedures for measuring drug response described here were developed by the NIH LINCS program to improve reproducibility. Key features include maximizing uniform cell growth during the assay period, accounting for the effects of cell density on response, and correcting sensitivity measures for differences in proliferation rates...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28253435/detecting-membrane-protein-protein-interactions-using-the-mammalian-membrane-two-hybrid-mamth-assay
#16
Punit Saraon, Ingrid Grozavu, Sang Hyun Lim, Jamie Snider, Zhong Yao, Igor Stagljar
Protein-protein interactions (PPIs) play an integral role in numerous cellular processes. Membrane protein interactions, in particular, are critical in cellular responses to stresses and stimuli, with dysfunction of these PPIs (e.g., due to aberrant expression and/or mutation of interaction partners) leading to a diverse array of pathological states. Exploration of the interaction space and dynamics of membrane proteins is difficult due to the limitations of current techniques used to study proteins in the biochemically complex environment of biological membranes...
March 2, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28253434/a-general-non-radioactive-atpase-assay-for-chromatin-remodeling-complexes
#17
Benjamin Z Stanton, Courtney Hodges, Gerald R Crabtree, Keji Zhao
Chromatin remodeling complexes couple the energy released from ATP hydrolysis to facilitate transcription, recombination, and repair mechanisms essential for a wide variety of biologic responses. While recombinant expression of the regulatory subunits of these enzymes is possible, measuring catalytic (ATPase) activity of the intact complexes recovered from normal or mutant cells is critical for understanding their mechanisms. SWI/SNF-like remodeling complexes can be megadaltons in size and include many regulatory subunits, making reconstitution of purified subunits challenging for recapitulating in vivo function...
March 2, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28253433/ubfluor-a-fluorescent-thioester-to-monitor-hect-e3-ligase-catalysis
#18
David T Krist, Peter K Foote, Alexander V Statsyuk
HECT E3 ubiquitin ligases (∼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1∼Ub, E2∼Ub, and HECT E3∼Ub (where ∼ indicates a thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes, UbFluor, that act as mechanistically relevant pseudosubstrates of HECT E3s...
March 2, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27925669/biochemical-biophysical-and-cellular-techniques-to-study-the-guanine-nucleotide-exchange-factor-giv-girdin
#19
Pradipta Ghosh, Nicolas Aznar, Lee Swanson, I-Chung Lo, Inmaculada Lopez-Sanchez, Jason Ear, Cristina Rohena, Nicholas Kalogriopoulos, Linda Joosen, Ying Dunkel, Nina Sun, Peter Nguyen, Deepali Bhandari
Canonical signal transduction via heterotrimeric G proteins is spatiotemporally restricted, i.e., triggered exclusively at the plasma membrane, only by agonist activation of G protein-coupled receptors via a finite process that is terminated within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a noncanonical pathway for activation of heterotrimeric G proteins via the nonreceptor guanidine-nucleotide exchange factor, GIV/Girdin. Biochemical, biophysical, and functional studies evaluating this pathway have unraveled its unique properties and distinctive spatiotemporal features...
December 7, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27925668/cyclic-immunofluorescence-cycif-a-highly-multiplexed-method-for-single-cell-imaging
#20
Jia-Ren Lin, Mohammad Fallahi-Sichani, Jia-Yun Chen, Peter K Sorger
Cyclic Immunofluorescence (CycIF) is a public-domain method for performing highly multiplexed immunofluorescence imaging using a conventional epifluorescence microscope. It uses simple reagents and existing antibodies to construct images with up to 30 channels by sequential 4- to 6-channel imaging followed by fluorophore inactivation. Three variant methods are described, the most generally useful of which involves staining fixed cells with antibodies directly conjugated to Alexa Fluor dyes and imaging in four colors, inactivating fluorophores using a mild base in the presence of hydrogen peroxide and light, and then performing another round of staining and imaging...
December 7, 2016: Current Protocols in Chemical Biology
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