987 results match your criteria solution-phase biological


Reaction Path-Force Matching in Collective Variables: Determining Ab Initio QM/MM Free Energy Profiles by Fitting Mean Force.

J Chem Theory Comput 2021 Jul 20. Epub 2021 Jul 20.

Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, 402 North Blackford Street, Indianapolis, Indiana 46202, United States.

First-principles determination of free energy profiles for condensed-phase chemical reactions is hampered by the daunting costs associated with configurational sampling on ab initio quantum mechanical/molecular mechanical (AI/MM) potential energy surfaces. Here, we report a new method that enables efficient AI/MM free energy simulations through mean force fitting. In this method, a free energy path in collective variables (CVs) is first determined on an efficient reactive aiding potential. Read More

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Spectroscopic and computational investigations of organometallic complexation of group 12 transition metals by methanobactins from Methylocystis sp. SB2.

J Inorg Biochem 2021 Jun 2;223:111496. Epub 2021 Jun 2.

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA. Electronic address:

Methanotrophic bacteria catalyze the aerobic oxidation of methane to methanol using Cu-containing enzymes, thereby exerting a modulating influence on the global methane cycle. To facilitate the acquisition of Cu ions, some methanotrophic bacteria secrete small modified peptides known as "methanobactins," which strongly bind Cu and function as an extracellular Cu recruitment relay, analogous to siderophores and Fe. In addition to Cu, methanobactins form complexes with other late transition metals, including the Group 12 transition metals Zn, Cd, and Hg, although the interplay among solution-phase configurations, metal interactions, and the spectroscopic signatures of methanobactin-metal complexes remains ambiguous. Read More

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Cyclic Peptoid-Peptide Hybrids as Versatile Molecular Transporters.

Front Chem 2021 25;9:696957. Epub 2021 Jun 25.

Institute of Biological and Chemical Systems- Functional Molecular Systems (IBCS-FMS), Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.

Addressing intracellular targets is a challenging task that requires potent molecular transporters capable to deliver various cargos. Herein, we report the synthesis of hydrophobic macrocycles composed of both amino acids and peptoid monomers. The cyclic tetramers and hexamers were assembled in a modular approach using solid as well as solution phase techniques. Read More

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An ultrasensitive homogeneous electrochemical biosensor based on CRISPR-Cas12a.

Anal Methods 2021 Jul 8. Epub 2021 Jul 8.

Key Laboratory for Analytical Science of Food Safety and Biology (MOE & Fujian Province), State Key Laboratory of Photocatalysis on Energy and Environment, Department of Chemistry, Fuzhou University, Fuzhou 350116, P. R. China.

Taking advantage of the high-efficiency indiscriminate ssDNA cleavage activity of Cas12a in combination with the diffusivity difference of methylene blue (MB)-labeled probes (short oligonucleotides/mononucleotides) toward the negatively-charged indium tin oxide (ITO) electrode, a simple, immobilization-free, highly sensitive, and homogeneous electrochemical biosensor for the detection of human papillomavirus (HPV-16) has been fabricated. At the core of the detection process, Cas12a employed ssDNA trans-cleavage capability to achieve short-strand nucleotide cleavage, while MB-labeled probes served as high-efficiency homogeneous electrochemical emitters to achieve differential pulse voltammetric (DPV) signal. Specifically, due to strong electrostatic repulsion, MB-labeled short oligonucleotides (reporter) cannot diffuse freely to the surface of the negatively charged ITO electrode, and only weak electrochemical signals can be detected. Read More

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Tuning Chromophore-Based LMOF Dimensionality to Enhance Detection Sensitivity for Fe Ions.

ACS Omega 2021 Jun 17;6(25):16498-16506. Epub 2021 Jun 17.

Department of Chemistry and Chemical Biology, Rutgers University, 123 Bevier Road, Piscataway, New Jersey 08854, United States.

Herein, we report the synthesis of two new manganese-based luminescent metal-organic frameworks (LMOFs) [Mn(tipe)(1,4-ndc)] () and [Mn(tipe)(1,4-ndc) (HO)·(DMF)·(HO)] () [tipe = 1,1,2,2-tetrakis(4-(1-imidazol-1-yl)phenyl)ethene (tipe) and 1,4-ndc = 1,4-naphthalenedicarboxylic acid] constructed from an aggregation-induced emission (AIE) chromophore ligand. Compound can undergo a facile single-crystal-to-single-crystal transformation to form compound , which results in an increase in dimensionality from a two-dimensional (2D) network to a three-dimensional (3D) network. Both compounds demonstrate excellent performance for the solution-phase detection of Fe ions through a significant and rapid quench in luminescence emission. Read More

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[Determination of the binding of natural products to the human c-myb oncogene promoter G-quadruplex DNA by capillary electrophoresis and electrospray ionization mass spectrometry].

Se Pu 2020 Sep;38(9):1069-1077

Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada.

The relationship between a drug and its target directly affects its pharmacology and efficacy. Drug-target binding ability and binding stoichiometry are essential characterization data in pharmaceutical research. The c-myb proto-oncogene encodes a crucial transcription factor that is involved in proliferation, differentiation, and maturation during hematopoiesis. Read More

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September 2020

Consistent inclusion of continuum solvation in energy decomposition analysis: theory and application to molecular CO reduction catalysts.

Chem Sci 2020 Nov 27;12(4):1398-1414. Epub 2020 Nov 27.

Department of Chemistry, University of California at Berkeley Berkeley CA 94720 USA

To facilitate computational investigation of intermolecular interactions in the solution phase, we report the development of ALMO-EDA(solv), a scheme that allows the application of continuum solvent models within the framework of energy decomposition analysis (EDA) based on absolutely localized molecular orbitals (ALMOs). In this scheme, all the quantum mechanical states involved in the variational EDA procedure are computed with the presence of solvent environment so that solvation effects are incorporated in the evaluation of its energy components. After validation on several model complexes, we employ ALMO-EDA(solv) to investigate substituent effects on two classes of complexes that are related to molecular CO reduction catalysis. Read More

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November 2020

Structure sampling for computational estimation of localized DNA interaction rates.

Sci Rep 2021 Jun 16;11(1):12730. Epub 2021 Jun 16.

Department of Computer Science, University of New Mexico, Albuquerque, NM, 87131, USA.

Molecular circuits implemented using molecular components tethered to a DNA tile nanostructure have certain advantages over solution-phase circuits. Tethering components in close proximity increases the speed of reactions by reducing diffusion and improves scalability by enabling reuse of identical DNA sequences at different locations in the circuit. These systems show great potential for practical applications including delivery of diagnostic and therapeutic molecular circuits to cells. Read More

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Direct Mapping of Higher-Order RNA Interactions by SHAPE-JuMP.

Biochemistry 2021 Jun 14;60(25):1971-1982. Epub 2021 Jun 14.

Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States.

Higher-order structure governs function for many RNAs. However, discerning this structure for large RNA molecules in solution is an unresolved challenge. Here, we present SHAPE-JuMP (selective 2'-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) to interrogate through-space RNA tertiary interactions. Read More

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Padlock probe-based rolling circle amplification lateral flow assay for point-of-need nucleic acid detection.

Analyst 2021 Jun;146(13):4340-4347

School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523, USA. and Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA.

Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs) such as PCR and isothermal amplification methods provide excellent analytical performance and significantly faster turnaround times than conventional culture-based methods. However, the inherent cost and complexity of NAATs limit their application in resource-limited settings and the developing world. Read More

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Macrocycle synthesis strategy based on step-wise "adding and reacting" three components enables screening of large combinatorial libraries.

Chem Sci 2020 Jun 26;11(30):7858-7863. Epub 2020 Jun 26.

Institute of Chemical Sciences and Engineering, School of Basic Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL) CH-1015 Lausanne Switzerland

Macrocycles provide an attractive modality for drug development, but generating ligands for new targets is hampered by the limited availability of large macrocycle libraries. We have established a solution-phase macrocycle synthesis strategy in which three building blocks are coupled sequentially in efficient alkylation reactions that eliminate the need for product purification. We demonstrate the power of the approach by combinatorially reacting 15 bromoacetamide-activated tripeptides, 42 amines, and 6 bis-electrophile cyclization linkers to generate a 3780-compound library with minimal effort. Read More

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Antibody-receptor interactions mediate antibody-dependent cellular cytotoxicity.

J Biol Chem 2021 May 24;297(1):100826. Epub 2021 May 24.

Protein Analytical Chemistry Department, Genentech Inc, South San Francisco, California, USA. Electronic address:

Binding of antibodies to their receptors is a core component of the innate immune system. Understanding the precise interactions between antibodies and their Fc receptors has led to the engineering of novel mAb biotherapeutics with tailored biological activities. One of the most significant findings is that afucosylated monoclonal antibodies demonstrate increased affinity toward the receptor FcγRIIIa, with a commensurate increase in antibody-dependent cellular cytotoxicity. Read More

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Laser Photodissociation Action Spectroscopy for the Wavelength-Dependent Evaluation of Photoligation Reactions.

Anal Chem 2021 06 21;93(22):8091-8098. Epub 2021 May 21.

Central Analytical Research Facility, Queensland University of Technology (QUT), 2 George Street, Brisbane, QLD 4000, Australia.

The nitrile imine-mediated tetrazole-ene cycloaddition is a widely used class of photoligation. Optimizing the reaction outcome requires detailed knowledge of the tetrazole photoactivation profile, which can only partially be ascertained from absorption spectroscopy, or otherwise involves laborious reaction monitoring in solution. Photodissociation action spectroscopy (PDAS) combines the advantages of optical spectroscopy and mass spectrometry in that only absorption events resulting in a mass change are recorded, thus revealing the desired wavelength dependence of product formation. Read More

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DNA walker-mediated biosensor for target-triggered triple-mode detection of Vibrio parahaemolyticus.

Biosens Bioelectron 2021 May 7;186:113305. Epub 2021 May 7.

State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, State Key Laboratory Base of Novel Functional Materials and Preparation Science, School of Materials Science and Chemical Engineering, Ningbo University, Ningbo, 315211, PR China.

Herein, we have constructed a target-triggered and DNA walker-mediated biosensor with triple signal (BTS) outputs mode for sensitive and reliable detection of pathogenic bacteria. Vibrio parahaemolyticus (VP) being the detection target model, the aptamer conformational changes induced by VP have been designed to activate the DNA walk on the modifiable and conductive surface of FeO nanoparticles to generate triple signal outputs, including electrochemiluminescence (ECL), fast scan cyclic voltammetry (FSCV) and fluorescent pixel counting (FLPC). Limits of quantification (LOQ) of VP were as low as 1 CFU⋅mL by ECL with a linear range of 1-10 CFU⋅mL, 1 CFU⋅mL by FSCV with a linear range of 1-10 CFU⋅mL, and 10 CFU⋅mL by FLPC with a linear range of 10-10 CFU⋅mL respectively, all squared correlation coefficients R being > 0. Read More

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Surface-Induced Dissociation of Anionic vs Cationic Native-Like Protein Complexes.

J Am Chem Soc 2021 May 13;143(20):7698-7706. Epub 2021 May 13.

Department of Chemistry and Biochemistry and Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State University, Columbus, Ohio 43210, United States.

Characterizing protein-protein interactions, stoichiometries, and subunit connectivity is key to understanding how subunits assemble into biologically relevant, multisubunit protein complexes. Native mass spectrometry (nMS) has emerged as a powerful tool to study protein complexes due to its low sample consumption and tolerance for heterogeneity. In nMS, positive mode ionization is routinely used and charge reduction, through the addition of solution additives, is often used, as the resulting lower charge states are often considered more native-like. Read More

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A MATLAB toolbox for modeling genetic circuits in cell-free systems.

Synth Biol (Oxf) 2021 5;6(1):ysab007. Epub 2021 Feb 5.

Control and Dynamical Systems and Biology and Biological Engineering, California Institute of Technology, 1200 E California Blvd, 91125, CA, USA.

We introduce a MATLAB-based simulation toolbox, called txtlsim, for an based Transcription-Translation (TX-TL) system. This toolbox accounts for several cell-free-related phenomena, such as resource loading, consumption and degradation, and in doing so, models the dynamics of TX-TL reactions for the entire duration of solution phase batch-mode experiments. We use a Bayesian parameter inference approach to characterize the reaction rate parameters associated with the core transcription, translation and mRNA degradation mechanics of the toolbox, allowing it to reproduce constitutive mRNA and protein-expression trajectories. Read More

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February 2021

Non-thermal plasma-induced DMPO-OH yields hydrogen peroxide.

Arch Biochem Biophys 2021 Jul 6;705:108901. Epub 2021 May 6.

Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Showa-Ku, Nagoya, 466-8550, Japan; Center for Low-temperature Plasma Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8603, Japan. Electronic address:

Recent developments in electronics have enabled the medical applications of non-thermal plasma (NTP), which elicits reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydroxyl radical (OH), hydrogen peroxide (HO), singlet oxygen (O), superoxide (O), ozone, and nitric oxide at near-physiological temperatures. In preclinical studies or human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral and biofilm-related infections, wound healing, and cancer cell death. To elucidate the solution-phase biological effects of NTP in the presence of biocompatible reducing agents, we employed electron paramagnetic resonance (EPR) spectroscopy to quantify OH using a spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); O using a fluorescent probe; and O and HO using luminescent probes in the presence of thiols or tempol. Read More

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Faster Surface Ligation Reactions Improve Immobilized Enzyme Structure and Activity.

J Am Chem Soc 2021 May 29;143(18):7154-7163. Epub 2021 Apr 29.

Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, United States.

During integration into materials, the inactivation of enzymes as a result of their interaction with nanometer size denaturing "hotspots" on surfaces represents a critical challenge. This challenge, which has received far less attention than improving the long-term stability of enzymes, may be overcome by limiting the exploration of surfaces by enzymes. One way this may be accomplished is through increasing the rate constant of the surface ligation reaction and thus the probability of immobilization with reactive surface sites (i. Read More

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Recent Advances on the Selection Methods of DNA-Encoded Libraries.

Chembiochem 2021 Jul 7;22(14):2384-2397. Epub 2021 May 7.

Department of Chemistry and the State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.

DNA-encoded libraries (DEL) have come of age and become a major technology platform for ligand discovery in both academia and the pharmaceutical industry. Technological maturation in the past two decades and the recent explosive developments of DEL-compatible chemistries have greatly improved the chemical diversity of DELs and fueled its applications in drug discovery. A relatively less-covered aspect of DELs is the selection method. Read More

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Hybrid Polyoxometalate Salt Adhesion by Butyltin Functionalization.

ACS Appl Mater Interfaces 2021 Apr 15;13(16):19497-19506. Epub 2021 Apr 15.

Department of Chemistry, Oregon State University, Corvallis, Oregon 97331, United States.

Polyoxometalate (POM)-based ionic liquids, with nearly infinite compositional variations to fine-tune antimicrobial and physical properties, function as water purification filters, anticorrosion/antibacterial coatings for natural stones, self-repairing acid-resistant coatings, catalysts, and electroactive, stable solvents. By combining hydrophobic quaternary ammonium cations (QACs; tetraheptylammonium and trihexyltetradecylammonium) with butyltin-substituted polyoxotungstates [(BuSn)(α-SiWO)] via repeated solvent extraction-ion exchange, we obtained phase-pure hybrid POM salts (referred to as such because they melt above room temperature). If the solvent extraction process is performed only once, then solids with high salt contamination and considerably lower melting temperatures are obtained. Read More

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A Simple Method for Developing a Hand-Drawn Paper-Based Sensor for Mercury; Using Green Synthesized Silver Nanoparticles and Smartphone as a Hand-Held-Device for Colorimetric Assay.

Glob Chall 2021 Apr 15;5(4):2000099. Epub 2021 Feb 15.

Pharmaceutical Sciences Research Center Health institute Kermanshah University of Medical Sciences Kermanshah 6715847141 Iran.

Mercury ions are highly toxic at trace levels, and its pollution has posed a significant threat to the environment and public health, where current detection methods mainly require laborious operation and expensive instrumentation. Herein, a simple, cost-effective, instrument-free approach for selective detection of Hg based on a hand-drawn paper-based naked-eye colorimetric device is developed. To develop a hand-drawn paper-based device, a crayon is used to build hydrophobic barriers and a paper puncher is applied to obtain patterns as a sensing zone. Read More

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Grand-Canonical Model of Electrochemical Double Layers from a Hybrid Density-Potential Functional.

J Chem Theory Comput 2021 Apr 31;17(4):2417-2430. Epub 2021 Mar 31.

Institute of Energy and Climate Research, Theory and Computation of Energy Materials (IEK-13), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.

A hybrid density-potential functional of an electrochemical interface that encompasses major effects in the contacting metal and electrolyte phases is formulated. Variational analysis of this functional yields a grand-canonical model of the electrochemical double layer (EDL). Specifically, metal electrons are described using the Thomas-Fermi-Dirac-Wigner theory of an inhomogeneous electron gas. Read More

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Chemical Reactivities of Two Widely Used Ruthenium-Based CO-Releasing Molecules with a Range of Biologically Important Reagents and Molecules.

Anal Chem 2021 03 21;93(12):5317-5326. Epub 2021 Mar 21.

Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30303, United States.

Ruthenium-based CO-releasing molecules (CO-RMs), CORM-2 and CORM-3, have been widely used as surrogates of CO for studying its biological effects and with much success. However, several previous solution-phase and studies have revealed the ability of such CO-RMs to chemically modify proteins and reduce aromatic nitro groups due to their intrinsic chemical reactivity under certain conditions. In our own work of studying the cytoprotective effects of CO donors, we were in need of assessing chemical factors that could impact the interpretation of results from CO donors including CORM-2,3 in various assays. Read More

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Synthesis of Novel Peptides Using Unusual Amino Acids.

Iran J Pharm Res 2020 ;19(3):370-382

Department of Environmental Sciences, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, Tehran, Iran.

Small peptides are valuable peptides due to their extended biological activities. Their activities could be categorized according to their low antigenicity, osmotic pressure, and also because of their astonishing bioactivities. For example, the aggression of Phe-Phe fibers via self-assembly and intermolecular hydrogen bonding is the main reason for the formation of Alzheimer's β-amyloid fibrils. Read More

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January 2020

Synthesis and Bioactivity of a Cyclopolypeptide from Caribbean Marine Sponge.

Iran J Pharm Res 2020 ;19(3):156-170

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, Semeling, Bedong, Kedah, Malaysia.

Synthesis of a natural proline-rich cyclopolypeptide - rolloamide A was carried out by coupling of tri- and tetrapeptide units Boc-Phe-Pro-Val-OMe and Boc-Pro-Leu-Pro-Ile-OMe after proper deprotection at carboxyl and amino terminals using carbodiimide chemistry in alkaline environment followed by cyclization of linear heptapeptide segment in the presence of base. The structure of synthesized peptide was confirmed by spectral techniques including FTIR, H NMR, C NMR, MS analyses. Newly synthesized peptide was subjected to biological screening against pathogenic microbes and earthworms. Read More

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January 2020

Mechanical coupling in the nitrogenase complex.

PLoS Comput Biol 2021 03 4;17(3):e1008719. Epub 2021 Mar 4.

Physical and Computational Sciences Directorate, Pacific Northwestern National Laboratory, Richland, Washington United States of America.

The enzyme nitrogenase reduces dinitrogen to ammonia utilizing electrons, protons, and energy obtained from the hydrolysis of ATP. Mo-dependent nitrogenase is a symmetric dimer, with each half comprising an ATP-dependent reductase, termed the Fe Protein, and a catalytic protein, known as the MoFe protein, which hosts the electron transfer P-cluster and the active-site metal cofactor (FeMo-co). A series of synchronized events for the electron transfer have been characterized experimentally, in which electron delivery is coupled to nucleotide hydrolysis and regulated by an intricate allosteric network. Read More

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Impact of Ambient Vapors Present in an Electrospray Ionization Source on Gas-Phase Ion Structures.

J Am Soc Mass Spectrom 2021 Mar 19;32(3):725-735. Epub 2021 Feb 19.

Center for Mass Spectrometry, Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, New Jersey 07030, United States.

According to current consensus, structures of protomeric (or deprotomeric) tautomers of gaseous ions generated by electrospray ionization depend primarily on the nature of the spray solvent. To probe the effect of the spray solvent on protonation, 4-aminobenzoic acid (PABA) has often been selected as the model compound. It is widely accepted that the protonation in the gas phase takes place primarily on the carbonyl oxygen atom when the sample is sprayed in methanol and on the nitrogen atom when acetonitrile is used as the spray solvent. Read More

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Structural Flexibility of Cyclosporine A Is Mediated by Amide - Isomerization and the Chameleonic Roles of Calcium.

J Phys Chem B 2021 02 1;125(5):1378-1391. Epub 2021 Feb 1.

Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996, United States.

Falling outside of Lipinski's rule of five, macrocyclic drugs have accessed unique binding sites of their target receptors unreachable by traditional small molecules. Cyclosporin(e) A (CycA), an extensively studied macrocyclic natural product, is an immunosuppressant with undesirable side effects such as electrolytic imbalances. In this work, a comprehensive view on the conformational landscape of CycA, its interactions with Ca, and host-guest interactions with cyclophilin A (CypA) is reported through exhaustive analyses that combine ion-mobility spectrometry-mass spectrometry (IMS-MS), nuclear magnetic resonance (NMR) spectroscopy, distance-geometry modeling, and NMR-driven molecular dynamics. Read More

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February 2021

SINGLE: Atomic-resolution structure identification of nanocrystals by graphene liquid cell EM.

Sci Adv 2021 Jan 29;7(5). Epub 2021 Jan 29.

Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia.

Analysis of the three-dimensional (3D) structures of nanocrystals with solution-phase transmission electron microscopy is beginning to reveal their unique physiochemical properties. We developed a "one-particle Brownian 3D reconstruction method" based on imaging of ensembles of colloidal nanocrystals using graphene liquid cell electron microscopy. Projection images of differently rotated nanocrystals are acquired using a direct electron detector with high temporal (<2. Read More

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January 2021

Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing.

Nucleic Acids Res 2021 04;49(7):e41

Department of Chemistry, University of Kansas, Lawrence, KS 66045, USA.

Modifications in RNA are numerous (∼170) and in higher numbers compared to DNA (∼5) making the ability to sequence an RNA molecule to identify these modifications highly tenuous using next generation sequencing (NGS). The ability to immobilize an exoribonuclease enzyme, such as XRN1, to a solid support while maintaining its activity and capability to cleave both the canonical and modified ribonucleotides from an intact RNA molecule can be a viable approach for single-molecule RNA sequencing. In this study, we report an enzymatic reactor consisting of covalently attached XRN1 to a solid support as the groundwork for a novel RNA exosequencing technique. Read More

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