RESUMEN

Riboswitches are segments of noncoding RNA that bind with metabolites, resulting in a change in gene expression. To understand the molecular mechanism of gene regulation in a fluoride riboswitch, a base-pair opening dynamics study was performed with and without ligands using the Bacillus cereus fluoride riboswitch. We demonstrate that the structural stability of the fluoride riboswitch is caused by two steps depending on ligands. Upon binding of a magnesium ion, significant changes in a conformation of the riboswitch occur, resulting in the greatest increase in their stability and changes in dynamics by a fluoride ion. Examining hydrogen exchange dynamics through NMR spectroscopy, we reveal that the stabilization of the U45·A37 base-pair due to the binding of the fluoride ion, by changing the dynamics while maintaining the structure, results in transcription regulation. Our results demonstrate that the opening dynamics and stabilities of a fluoride riboswitch in different ion states are essential for the genetic switching mechanism.

Asunto(s)

Bacillus cereus/genética , Emparejamiento Base , Fluoruros/química , Genes Bacterianos , Riboswitch , Aptámeros de Nucleótidos , Secuencia de Bases , Catálisis , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Motivos de Nucleótidos

RESUMEN

In order to utilize wave energy, various wave power systems are being actively researched and developed and interest in them is increasing. To maximize the operational efficiency, it is very important to monitor and maintain the fault of components of the system. In recent years, interest in the management cost, high reliability and facility utilization of such systems has increased. In this regard, fault diagnosis technology including fault factor analysis and fault reproduction is drawing attention as an important main technology. Therefore, in this study, to reproduce and monitor the faults of a wave power system, firstly, the failure mode of the system was analyzed using FMEA analysis. Secondly, according to the derived failure mode and effect, the thrust bearing was selected as a target for fault reproduction and a test equipment bench was constructed. Finally, with the vibration data obtained by conducting the tests, the vibration spectrum was analyzed to extract the features of the data for each operating status; the data was classified by applying the three machine learning algorithms: naïve Bayes (NB), k-nearest neighbor (k-NN), and multi-layer perceptron (MLP). The criteria for determining the fault were derived. It is estimated that a more efficient fault diagnosis is possible by using the standard and fault monitoring method of this study.

RESUMEN

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RESUMEN

STING, a central protein in the innate immune response to cytosolic DNA, has emerged as a hot target for the development of vaccine-adjuvants and anticancer drugs. The discovery of potent human-STING (hSTING) agonist is expected to revolutionize the current cancer immunotherapy. Inspired by the X-ray crystal structure of DMXAA (5,6-dimethylxanthenone-4-acetic acid) and hSTINGG230I complex, we designed various DMXAA derivatives that contain a hydrogen bonding donor/acceptor or a halide at the C7 position. While 7-bromo- and 7-hydroxyl-DMXAA showed notable binding to mouse-STING (mSTING), our newly synthesized C7-functionalized DMXAA derivatives did not bind to hSTING. Nevertheless, our newly developed synthetic protocol for the C7-functionalization of DMXAA would be applicable to access other C7-substituted DMXAA analogues as potential hSTING agonists.

Asunto(s)

Diseño de Fármacos , Proteínas de la Membrana/agonistas , Xantonas/farmacología , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Temperatura , Xantonas/síntesis química , Xantonas/química

RESUMEN

Dynamic ensembles hold great promise in advancing RNA-targeted drug discovery. Here we subjected the transactivation response element (TAR) RNA from human immunodeficiency virus type-1 to experimental high-throughput screening against ~100,000 drug-like small molecules. Results were augmented with 170 known TAR-binding molecules and used to generate sublibraries optimized for evaluating enrichment when virtually screening a dynamic ensemble of TAR determined by combining NMR spectroscopy data and molecular dynamics simulations. Ensemble-based virtual screening scores molecules with an area under the receiver operator characteristic curve of ~0.85-0.94 and with ~40-75% of all hits falling within the top 2% of scored molecules. The enrichment decreased significantly for ensembles generated from the same molecular dynamics simulations without input NMR data and for other control ensembles. The results demonstrate that experimentally determined RNA ensembles can significantly enrich libraries with true hits and that the degree of enrichment is dependent on the accuracy of the ensemble.

Asunto(s)

Descubrimiento de Drogas/métodos , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , ARN Viral/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular

RESUMEN

Retinoic acid-inducible gene I (RIG-I) recognizes double-stranded viral RNAs (dsRNAs) containing two or three 5' phosphates. A few reports of 5'-PPP-independent RIG-I agonists have emerged, but little is known about the molecular principles underlying their recognition. We recently found that the bent duplex RNA from the influenza A panhandle promoter activates RIG-I even in the absence of a 5'-triphosphate moiety. Here, we report that non-canonical synthetic RNA oligonucleotides containing G-U wobble base pairs that form a bent helix can exert RIG-I-mediated antiviral and anti-tumor effects in a sequence- and site-dependent manner. We present synthetic RNAs that have been systematically modified to enhance their efficacy and we outline the basic principles for engineering RIG-I agonists applicable to immunotherapy.

RESUMEN

Diversification of RNA-targeted scaffolds offers great promise in the search for selective ligands of therapeutically relevant RNA such as HIV-1 TAR. We herein report the establishment of amiloride as a novel RNA-binding scaffold along with synthetic routes for combinatorial C(5)- and C(6)-diversification. Iterative modifications at the C(5)- and C(6)- positions yielded derivative 24, which demonstrated a 100-fold increase in activity over the parent dimethylamiloride in peptide displacement assays. NMR chemical shift mapping was performed using the 2D SOFAST- [1H-13C] HMQC NMR method, which allowed for facile and rapid evaluation of binding modes for all library members. Cheminformatic analysis revealed distinct differences between selective and non-selective ligands. In this study, we evolved dimethylamiloride from a weak TAR ligand to one of the tightest binding selective TAR ligands reported to date through a novel combination of synthetic methods and analytical techniques. We expect these methods to allow for rapid library expansion and tuning of the amiloride scaffold for a range of RNA targets and for SOFAST NMR to allow unprecedented evaluation of small molecule:RNA interactions.

RESUMEN

Cyclic GMP-AMP synthase (cGAS) is a DNA-sensing enzyme in the innate immune system. Recent studies using core-cGAS lacking the N terminus investigated the mechanism for binding of double-stranded (ds) DNA and synthesis of 2',3'-cyclic GMP-AMP (cGAMP), a secondary messenger that ultimately induces type I interferons. However, the function of the N terminus of cGAS remains largely unknown. Here, we found that the N terminus enhanced the activity of core-cGAS in vivo. Importantly, the catalytic activity of core-cGAS decreased as the length of double-stranded DNA (dsDNA) increased, but the diminished activity was restored by addition of the N terminus. Furthermore, the N terminus de­oligomerized the 2 : 1 complex of core­cGAS and dsDNA into a 1 : 1 complex, suggesting that the N terminus enhanced the activity of core­cGAS by facilitating formation of a monomeric complex of cGAS and DNA.

Asunto(s)

ADN/química , Sustancias Macromoleculares/química , Nucleotidiltransferasas/química , Multimerización de Proteína , Animales , Biocatálisis , Calorimetría/métodos , Dicroismo Circular , ADN/genética , ADN/metabolismo , Humanos , Immunoblotting , Cinética , Sustancias Macromoleculares/metabolismo , Espectroscopía de Resonancia Magnética , Ratones , Nucleótidos Cíclicos/biosíntesis , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Unión Proteica

RESUMEN

Retinoic acid-inducible gene I (RIG-I) recognizes specific molecular patterns of viral RNAs for inducing type I interferon. The C-terminal domain (CTD) of RIG-I binds to double-stranded RNA (dsRNA) with the 5'-triphosphate (5'-PPP), which induces a conformational change in RIG-I to an active form. It has been suggested that RIG-I detects infection of influenza A virus by recognizing the 5'-triphosphorylated panhandle structure of the viral RNA genome. Influenza panhandle RNA has a unique structure with a sharp helical bending. In spite of extensive studies of how viral RNAs activate RIG-I, whether the structural elements of the influenza panhandle RNA confer the ability to activate RIG-I signaling has been poorly explored. Here, we investigated the dynamics of the influenza panhandle RNA in complex with RIG-I CTD using NMR spectroscopy and showed that the bending structure of the panhandle RNA negates the requirement of a 5'-PPP moiety for RIG-I activation.

Asunto(s)

Proteína 58 DEAD Box/metabolismo , Virus de la Influenza A/genética , Conformación de Ácido Nucleico , Polifosfatos/metabolismo , ARN Viral/química , Emparejamiento Base , Calorimetría , Proteína 58 DEAD Box/química , Humanos , Hidrógeno/metabolismo , Interferón beta/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Unión Proteica , Dominios Proteicos , Estabilidad del ARN , ARN Viral/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Inmunológicos , Termodinámica

RESUMEN

Z-DNA binding proteins (ZBPs) play important roles in RNA editing, innate immune response and viral infection. Structural and biophysical studies show that ZBPs initially form an intermediate complex with B-DNA for B-Z conversion. However, a comprehensive understanding of the mechanism of Z-DNA binding and B-Z transition is still lacking, due to the absence of structural information on the intermediate complex. Here, we report the solution structure of the Zα domain of the ZBP-containing protein kinase from Carassius auratus(caZαPKZ). We quantitatively determined the binding affinity of caZαPKZ for both B-DNA and Z-DNA and characterized its B-Z transition activity, which is modulated by varying the salt concentration. Our results suggest that the intermediate complex formed by caZαPKZ and B-DNA can be used as molecular ruler, to measure the degree to which DNA transitions to the Z isoform.

Asunto(s)

ADN Forma B/química , ADN de Forma Z/química , Proteínas de Unión al ADN/química , Proteínas de Peces/química , Carpa Dorada/metabolismo , Proteínas Quinasas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , ADN Forma B/metabolismo , ADN de Forma Z/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Expresión Génica , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Cloruro de Sodio/química , Termodinámica

RESUMEN

Higher sensitivity of NMR spectrometers and novel isotopic labeling schemes have ushered the development of rapid data acquisition methodologies, improving the time resolution with which NMR data can be acquired. For nucleic acids, longitudinal relaxation optimization in conjunction with Ernst angle excitation (SOFAST-HMQC) for imino protons, in addition to rendering rapid pulsing, has been demonstrated to yield significant improvements in sensitivity per unit time. Extending such methodology to other spins offers a viable prospect to measure additional chemical shifts, thereby broadening their utilization for various applications. Here, we introduce the 2D [(13)C, (1)H] aromatic SOFAST-HMQC that results in overall sensitivity gain of 1.4- to 1.7-fold relative to the conventional HMQC and can also be extended to yield long-range heteronuclear chemical shifts such as the adenine imino nitrogens N1, N3, N7 and N9. The applications of these experiments range from monitoring real-time biochemical processes, drug/ligand screening, and to collecting data at very low sample concentration and/or in cases where isotopic enrichment cannot be achieved.

Asunto(s)

Espectroscopía de Resonancia Magnética con Carbono-13/métodos , Resonancia Magnética Nuclear Biomolecular , Ácidos Nucleicos/química , Espectroscopía de Protones por Resonancia Magnética/métodos

RESUMEN

Using on- and off-resonance carbon and nitrogen R1ρ NMR relaxation dispersion in concert with mutagenesis and NMR chemical shift fingerprinting, we show that the transactivation response element RNA from the HIV-1 exists in dynamic equilibrium with a transient state that has a lifetime of ∼2 ms and population of ∼0.4%, which simultaneously remodels the structure of a bulge, stem, and apical loop. This is accomplished by a global change in strand register, in which bulge residues pair up with residues in the upper stem, causing a reshuffling of base pairs that propagates to the tip of apical loop, resulting in the creation of three noncanonical base pairs. Our results show that transient states can remodel distant RNA motifs and possibly give rise to mechanisms for rapid long-range communication in RNA that can be harnessed in processes such as cooperative folding and ribonucleoprotein assembly.

Asunto(s)

Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Conformación de Ácido Nucleico , ARN Viral/genética , Emparejamiento Base , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Termodinámica

RESUMEN

Dimethyl sulfoxide (DMSO) is widely used as a cosolvent to solubilize hydrophobic compounds in RNA-ligand binding assays. Although it is known that high concentrations of DMSO (>75%) can significantly affect RNA structure and folding energetics, a thorough analysis of how lower concentrations (<10%) of DMSO typically used in binding assays affects RNA structure and ligand binding has not been undertaken. Here, we use NMR and 2-aminopurine fluorescence spectroscopy to examine how DMSO affects the structure, dynamics, and ligand binding properties of two flexible hairpin RNAs: the transactivation response element from HIV-1 and bacterial ribosomal A-site. In both cases, 5-10% DMSO decreased stacking interactions and increased local disorder in noncanonical residues within bulges and loops and resulted in 0.3-4-fold reduction in the measured binding affinities for different small molecules, with the greatest reduction observed for an intercalating compound that binds RNA nonspecifically. Our results suggest that, by competing for hydrophobic interactions, DMSO can have a small but significant effect on RNA structure and ligand binding. These effects should be considered when developing ligand binding assays and high throughput screens.

Asunto(s)

Dimetilsulfóxido/farmacología , ARN/química , ARN/metabolismo , Secuencia de Bases , Evaluación Preclínica de Medicamentos , VIH-1/genética , Secuencias Invertidas Repetidas , Ligandos , ARN/genética , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Elementos de Respuesta , Ribosomas/genética

RESUMEN

Current approaches used to identify protein-binding small molecules are not suited for identifying small molecules that can bind emerging RNA drug targets. By docking small molecules onto an RNA dynamic ensemble constructed by combining NMR spectroscopy and computational molecular dynamics, we virtually screened small molecules that target the entire structure landscape of the transactivation response element (TAR) from HIV type 1 (HIV-1). We quantitatively predict binding energies for small molecules that bind different RNA conformations and report the de novo discovery of six compounds that bind TAR with high affinity and inhibit its interaction with a Tat peptide in vitro (K(i) values of 710 nM-169 µM). One compound binds HIV-1 TAR with marked selectivity and inhibits Tat-mediated activation of the HIV-1 long terminal repeat by 81% in T-cell lines and HIV replication in an HIV-1 indicator cell line (IC(50) ∼23.1 µM).

Asunto(s)

Descubrimiento de Drogas/métodos , ARN/metabolismo , Biología Computacional , VIH-1/efectos de los fármacos , Modelos Moleculares , Estructura Molecular , Netilmicina/farmacología , Unión Proteica , Replicación Viral/efectos de los fármacos