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https://www.readbyqxmd.com/read/29136199/site-specific-dual-color-labeling-of-long-rnas-for-single-molecule-spectroscopy
#1
Meng Zhao, Fabio D Steffen, Richard Börner, Michelle F Schaffer, Roland K O Sigel, Eva Freisinger
Labeling of long RNA molecules in a site-specific yet generally applicable manner is integral to many spectroscopic applications. Here we present a novel covalent labeling approach that is site-specific and scalable to long intricately folded RNAs. In this approach, a custom-designed DNA strand that hybridizes to the RNA guides a reactive group to target a preselected adenine residue. The functionalized nucleotide along with the concomitantly oxidized 3'-terminus can subsequently be conjugated to two different fluorophores via bio-orthogonal chemistry...
November 9, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29128539/injectable-nanocomposite-cryogels-for-versatile-protein-drug-delivery
#2
Sandeep T Koshy, David K Y Zhang, Joshua M Grolman, Alexander G Stafford, David J Mooney
Sustained, localized protein delivery can enhance the safety and activity of protein drugs in diverse disease settings. While hydrogel systems are widely studied as vehicles for protein delivery, they often suffer from rapid release of encapsulated cargo, leading to a narrow duration of therapy, and protein cargo can be denatured by incompatibility with the hydrogel crosslinking chemistry. In this work, we describe injectable nanocomposite hydrogels that are capable of sustained, bioactive, release of a variety of encapsulated proteins...
November 8, 2017: Acta Biomaterialia
https://www.readbyqxmd.com/read/29066549/selective-proteomic-analysis-of-antibiotic-tolerant-cellular-subpopulations-in-pseudomonas-aeruginosa-biofilms
#3
Brett M Babin, Lydia Atangcho, Mark B van Eldijk, Michael J Sweredoski, Annie Moradian, Sonja Hess, Tim Tolker-Nielsen, Dianne K Newman, David A Tirrell
Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics...
October 24, 2017: MBio
https://www.readbyqxmd.com/read/29043774/augmenting-influenza-specific-t-cell-memory-generation-with-a-natural-killer-t-cell-dependent-glycolipid-peptide-vaccine
#4
Regan J Anderson, Jasmine Li, Lukasz Kedzierski, Benjamin J Compton, Colin M Hayman, Taryn L Osmond, Ching-Wen Tang, Kathryn J Farrand, Hui-Fern Koay, Catarina Filipa Dos Santos Sa E Almeida, Lauren R Holz, Geoffrey M Williams, Margaret A Brimble, Zhongfang Wang, Marios Koutsakos, Katherine Kedzierska, Dale I Godfrey, Ian F Hermans, Stephen J Turner, Gavin F Painter
The development of a universal vaccine for influenza A virus (IAV) that does not require seasonal modification is a long-standing health goal, particularly in the context of the increasing threat of new global pandemics. Vaccines that specifically induce T cell responses are of considerable interest because they can target viral proteins that are more likely to be shared between different virus strains and subtypes and hence provide effective cross-reactive IAV immunity. From a practical perspective, such vaccines should induce T cell responses with long-lasting memory, while also being simple to manufacture and cost-effective...
November 17, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/29040874/surface-functionalized-exosomes-as-targeted-drug-delivery-vehicles-for-cerebral-ischemia-therapy
#5
Tian Tian, Hui-Xin Zhang, Chun-Peng He, Song Fan, Yan-Liang Zhu, Cui Qi, Ning-Ping Huang, Zhong-Dang Xiao, Zu-Hong Lu, Bakhos A Tannous, Jun Gao
The safe and effective delivery of drugs is a major obstacle in the treatment of ischemic stroke. Exosomes hold great promise as an endogenous drug delivery nanosystem for the treatment of cerebral ischemia given their unique properties, including low immunogenicity, innate stability, high delivery efficiency, and ability to cross the blood-brain barrier (BBB). However, exosome insufficient targeting capability limits their clinical applications. In this study, the c(RGDyK) peptide has been conjugated to the exosome surface by an easy, rapid, and bio-orthogonal chemistry...
October 4, 2017: Biomaterials
https://www.readbyqxmd.com/read/28989610/controlled-in-cell-activation-of-rna-therapeutics-using-bond-cleaving-bio-orthogonal-chemistry
#6
Irfan Khan, Leah M Seebald, Neil M Robertson, Mehmet V Yigit, Maksim Royzen
Temporal control of siRNA activation is a major challenge for RNAi-based therapeutics. The majority of the reported siRNA delivery systems rely on environmental factors, such as differences in extracellular and intracellular redox potential, ATP concentration, or pH to activate an siRNA payload. However dynamic endogenous environments are far too complex to rely on for controllable siRNA release and can result in premature siRNA activation prior to reaching the intended biological target. In addition, there are uncertainties about timing, degree and rate of the siRNA activation with spontaneous release approaches...
August 1, 2017: Chemical Science
https://www.readbyqxmd.com/read/28967990/click-chemistry-for-analysis-of-cell-proliferation-in-flow-cytometry
#7
Scott T Clarke, Veronica Calderon, Jolene A Bradford
The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology, but also in defining cellular responses to drug treatments. Changes in DNA replication during S-phase can provide valuable insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation is the incorporation of a thymidine analog during DNA synthesis...
October 2, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28935952/application-of-bio-orthogonal-proteome-labeling-to-cell-transplantation-and-heterochronic-parabiosis
#8
Yan Liu, Michael J Conboy, Melod Mehdipour, Yutong Liu, Thanhtra P Tran, Aaron Blotnick, Prasanna Rajan, Thalie Cavalcante Santos, Irina M Conboy
Studies of heterochronic parabiosis demonstrated that with age, the composition of the circulatory milieu changes in ways that broadly inhibit tissue regenerative capacity. In addition, local tissue niches have age-specific influences on their resident stem cells. Here we use bio-orthogonal proteome labeling for detecting in vivo proteins present only in transplanted myoblasts, but not in host tissue, and proteins exclusive to one young mouse and transferred during parabiosis to its old partner. We use a transgenic mouse strain that ubiquitously expresses a modified tRNA methionine synthase, metRS, which preferentially incorporates the methionine surrogate azido-nor-leucine (ANL) into newly generated proteins...
September 21, 2017: Nature Communications
https://www.readbyqxmd.com/read/28934470/monitoring-replication-protein-a-rpa-dynamics-in-homologous-recombination-through-site-specific-incorporation-of-non-canonical-amino-acids
#9
Nilisha Pokhrel, Sofia Origanti, Eric Parker Davenport, Disha Gandhi, Kyle Kaniecki, Ryan A Mehl, Eric C Greene, Chris Dockendorff, Edwin Antony
An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA...
September 19, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28751378/discovery-of-a-microbial-transglutaminase-enabling-highly-site-specific-labeling-of-proteins
#10
Wojtek Steffen, Fu Chong Ko, Jigar Patel, Victor Lyamichev, Thomas J Albert, Jörg Benz, Markus G Rudolph, Frank Bergmann, Thomas Streidl, Peter Kratzsch, Mara Boenitz-Dulat, Tobias Oelschlaegel, Michael Schraeml
Microbial transglutaminases (MTGs) catalyze the formation of Gln-Lys isopeptide bonds and are widely used for the cross-linking of proteins and peptides in food and biotechnological applications (e.g. to improve the texture of protein-rich foods or in generating antibody-drug conjugates). Currently used MTGs have low substrate specificity, impeding their biotechnological use as enzymes that do not cross-react with nontarget substrates (i.e. as bio-orthogonal labeling systems). Here, we report the discovery of an MTG from Kutzneria albida (KalbTG), which exhibited no cross-reactivity with known MTG substrates or commonly used target proteins, such as antibodies...
September 22, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28746829/imaging-chemistry-inside-living-cells-by-stimulated-raman-scattering-microscopy
#11
REVIEW
Hyeon Jeong Lee, Ji-Xin Cheng
Stimulated Raman scattering (SRS) microscopy is a vibrational imaging platform developed to visualize chemical content of a biological sample based on molecular vibrational fingerprints. With high-speed, high-sensitivity, and three-dimensional sectioning capability, SRS microscopy has been used to study chemical distribution, molecular transport, and metabolic conversion in living cells in a label-free manner. Moreover, aided with bio-orthogonal small-volume Raman probes, SRS microscopy allows direct imaging of metabolic activities of small molecules in living cells...
September 1, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28727448/extensive-survey-of-antibody-invariant-positions-for-efficient-chemical-conjugation-using-expanded-genetic-codes
#12
Akifumi Kato, Mitsuo Kuratani, Tatsuo Yanagisawa, Kazumasa Ohtake, Akiko Hayashi, Yoshimi Amano, Kaname Kimura, Shigeyuki Yokoyama, Kensaku Sakamoto, Yasuhisa Shiraishi
The site-specific chemical conjugation of proteins, following synthesis with an expanded genetic code, promises to advance antibody-based technologies, including antibody drug conjugation and the creation of bispecific Fab dimers. The incorporation of non-natural amino acids into antibodies not only guarantees site specificity but also allows the use of bio-orthogonal chemistry. However, the efficiency of amino acid incorporation fluctuates significantly among different sites, thereby hampering the identification of useful conjugation sites...
August 16, 2017: Bioconjugate Chemistry
https://www.readbyqxmd.com/read/28694021/recombinant-and-chemo-bio-orthogonal-synthesis-of-liposomal-thrombomodulin-and-its-antithrombotic-activity
#13
Lin Wang, Rui Jiang, Yang Liu, Maosheng Cheng, Qingyu Wu, Xue-Long Sun
Thrombomodulin (TM) is an endothelial cell membrane protein that acts as a major cofactor in the protein C anticoagulant pathway. The EGF-like domains 4-6 of TM (TM456) are essential for PC activation. In this study, we proposed a liposomal recombinant TM conjugate to mimic the membrane TM structure and its anticoagulant activity. First, a DSPE-PEG2000-TM456 was successfully synthesized by site-specific conjugation of azido-TM456 with DSPE-PEG2000-DBCO via copper-free click chemistry quantitatively. Then, liposome-TM456 was fabricated via direct liposome formation with the DSPE-PEG2000-TM456 and other lipids...
October 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28645043/in-vitro-and-in-vivo-behavior-of-dna-tetrahedrons-as-tumor-targeting-nanocarriers-for-doxorubicin-delivery
#14
Ji Hee Kang, Kyoung-Ran Kim, Hyukjin Lee, Dae-Ro Ahn, Young Tag Ko
Deoxyribonucleic acid (DNA) is a versatile material with high applicability and inherent biocompatibility. L-DNA, the perfect mirror form of the naturally occurring D-DNA, has been used in DNA nanotechnology. It has thermodynamically identical properties to D-DNA, is capable of self-assembly and bio-orthogonal base-pairing, and is resistant to nuclease activity. We previously constructed an L-DNA tetrahedron (L-Td) and found that this nanostructure has remarkably higher capacity for cell penetration than its natural counterpart (D-Td)...
September 1, 2017: Colloids and Surfaces. B, Biointerfaces
https://www.readbyqxmd.com/read/28598594/tethering-growth-factors-to-collagen-surfaces-using-copper-free-click-chemistry-surface-characterization-and-in-vitro-biological-response
#15
Hyun Jong Lee, Gabriella M Fernandes-Cunha, Ilham Putra, Won-Gun Koh, David Myung
Surface modifications with tethered growth factors have mainly been applied to synthetic polymeric biomaterials in well-controlled, acellular settings, followed by seeding with cells. The known bio-orthogonality of copper-free click chemistry provides an opportunity to not only use it in vitro to create scaffolds or pro-migratory tracks in the presence of living cells, but also potentially apply it to living tissues directly as a coupling modality in situ. In this study, we studied the chemical coupling of growth factors to collagen using biocompatible copper-free click chemistry and its effect on the enhancement of growth factor activity in vitro...
July 19, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28573212/bio-orthogonal-mediated-nucleic-acid-transfection-of-cells-via-cell-surface-engineering
#16
Paul J O'Brien, Sina Elahipanah, Dmitry Rogozhnikov, Muhammad N Yousaf
The efficient delivery of foreign nucleic acids (transfection) into cells is a critical tool for fundamental biomedical research and a pillar of several biotechnology industries. There are currently three main strategies for transfection including reagent, instrument, and viral based methods. Each technology has significantly advanced cell transfection; however, reagent based methods have captured the majority of the transfection market due to their relatively low cost and ease of use. This general method relies on the efficient packaging of a reagent with nucleic acids to form a stable complex that is subsequently associated and delivered to cells via nonspecific electrostatic targeting...
May 24, 2017: ACS Central Science
https://www.readbyqxmd.com/read/28524847/an-in-vivo-strategy-to-counteract-post-administration-anticoagulant-activity-of-azido-warfarin
#17
Sylvain Ursuegui, Marion Recher, Wojciech Krężel, Alain Wagner
Drugs, usually long acting and metabolically stable molecules, might be the source of adverse effects triggered by complex drug interactions, anaphylaxis and drug-induced coagulopathy. To circumvent this growing drug safety issue, we herein investigate the opportunity offered by bio-orthogonal chemistry for in vivo drug neutralization. We design a small-molecule anticoagulant drug (Warfarin) containing an azide group that acts as a safety pin. It allows drug deactivation and restoration of physiological coagulation via in vivo click reaction with a suitable cyclooctyne-based neutralizing agent...
May 19, 2017: Nature Communications
https://www.readbyqxmd.com/read/28500031/labeling-glycans-on-living-cells-by-a-chemoenzymatic-glycoengineering-approach
#18
Ruben T Almaraz, Yanhong Li
Structural glycobiology has traditionally been a challenging field due to a limited set of tools available to investigate the diverse and complex glycan molecules. However, we cannot ignore that glycans play critical roles in health as well as in disease, and are present in more than 50% of all proteins and on over 80% of all surface proteins. Chemoenzymatic glycoengineering (CGE) methods are a powerful set of tools to synthesize complex glycans, but the full potential of these methods have not been explored in cell biology yet...
June 15, 2017: Biology Open
https://www.readbyqxmd.com/read/28494698/extemporaneously-preparative-biodegradable-injectable-polymer-systems-exhibiting-temperature-responsive-irreversible-gelation
#19
Yasuyuki Yoshida, Kazuyuki Takata, Hiroki Takai, Keisuke Kawahara, Akinori Kuzuya, Yuichi Ohya
On clinical application of biodegradable injectable polymer (IP) systems, quick extemporaneous preparation of IP formulations and longer duration time gel state after injection into the body are the important targets to be developed. Previously, we had reported temperature-responsive covalent gelation systems via bio-orthogonal thiol-ene reaction by 'mixing strategy' of amphiphilic biodegradable tri-block copolymer (tri-PCG) attaching acryloyl groups on both termini (tri-PCG-Acryl) with reactive polythiol. In other previous works, we found 'freeze-dry with PEG/dispersion' method as quick extemporaneous preparation method of biodegradable IP formulations...
May 23, 2017: Journal of Biomaterials Science. Polymer Edition
https://www.readbyqxmd.com/read/28467039/zwitterionic-silane-copolymer-for-ultra-stable-and-bright-biomolecular-probes-based-on-fluorescent-quantum-dot-nanoclusters
#20
Fatimata Dembele, Mariana Tasso, Laura Trapiella-Alfonso, Xiangzhen Xu, Mohamed Hanafi, Nicolas Lequeux, Thomas Pons
Fluorescent semiconductor quantum dots (QDs) exhibit several unique properties that make them suitable candidates for biomolecular sensing, including high brightness, photostability, broad excitation, and narrow emission spectra. Assembling these QDs into robust and functionalizable nanosized clusters (QD-NSCs) can provide fluorescent probes that are several orders of magnitude brighter than individual QDs, thus allowing an even greater sensitivity of detection with simplified instrumentation. However, the formation of compact, antifouling, functionalizable, and stable QD-NSCs remains a challenging task, especially for a use at ultralow concentrations for single-molecule detection...
May 17, 2017: ACS Applied Materials & Interfaces
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