RESUMEN
RNA molecules are essential for carrying genetic information and regulating gene expression in most organisms including human pathogenic RNA and relate retro viruses. Targeting viral RNA (vRNA) structures provide broad opportunities to develop chemical tools to probe molecular virology and to discover novel targets for therapeutic intervention. An increasing number of RNA binding small molecules are being identified, stimulating increased interests in small molecule drug discovery for RNA targets. In this chapter, we describe protocols to characterize and robustly validate vRNA-small molecule (vRNA-sm) interactions starting from vRNA sample preparation, followed by small molecule screening against vRNA targets and finally to validating the vRNA-sm interactions via NMR spectroscopy and calorimetric titrations.
Asunto(s)
ARN Viral , Biofisica , Calorimetría , Humanos , ARN Viral/químicaRESUMEN
Alternative splicing of the HIV transcriptome is controlled through cis regulatory elements functioning as enhancers or silencers depending on their context and the type of host RNA binding proteins they recruit. Splice site acceptor A3 (ssA3) is one of the least used acceptor sites in the HIV transcriptome and its activity determines the levels of tat mRNA. Splice acceptor 3 is regulated by a combination of cis regulatory sequences, auxiliary splicing factors, and presumably RNA structure. The mechanisms by which these multiple regulatory components coordinate to determine the frequency in which ssA3 is utilized is poorly understood. By NMR spectroscopy and phylogenetic analysis, we show that the ssA3 regulatory locus is conformationally heterogeneous and that the sequences that encompass the locus are conserved across most HIV isolates. Despite the conformational heterogeneity, the major stem loop (A3SL1) observed in vitro folds to base pair the Polypyrimdine Tract (PPyT) to the Exon Splicing Silencer 2p (ESS2p) element and to a conserved downstream linker. The 3D structure as determined by NMR spectroscopy further reveals that the A3 consensus cleavage site is embedded within a unique stereochemical environment within the apical loop, where it is surrounded by alternating base-base interactions. Despite being described as a receptor for hnRNP H, the ESS2p element is sequestered by base pairing to the 3' end of the PPyT and within this context it cannot form a stable complex with hnRNP H. By comparison, hnRNP A1 directly binds to the A3 consensus cleavage site located within the apical loop, suggesting that it can directly modulate U2AF assembly. Sequence mutations designed to destabilize the PPyT:ESS2p helix results in an increase usage of ssA3 within HIV-infected cells, consistent with the PPyT becoming more accessible for U2AF recognition. Additional mutations introduced into the downstream ESS2 element synergize with ESS2p to cause further increases in ssA3 usage. When taken together, our work provides a unifying picture by which cis regulatory sequences, splicing auxiliary factors and RNA structure cooperate to provide stringent control over ssA3. We describe this as the pair-and-lock mechanism to restrict access of the PPyT, and posit that it operates to regulate a subset of the heterogenous structures encompassing the ssA3 regulatory locus.
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Empalme Alternativo , Infecciones por VIH , VIH-1 , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H , Sitios de Empalme de ARN , Factores de Empalme de ARN , ARN Viral , Secuencias Reguladoras de Ácido Ribonucleico , Infecciones por VIH/virología , VIH-1/genética , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Humanos , Mutación , Conformación de Ácido Nucleico , Factores de Empalme de ARN/metabolismo , ARN Viral/química , ARN Viral/metabolismoRESUMEN
Discoveries of RNA roles in cellular physiology and pathology are increasing the need for new tools that modulate the structure and function of these biomolecules, and small molecules are proving useful. In 2017, we curated the RNA-targeted BIoactive ligaNd Database (R-BIND) and discovered distinguishing physicochemical properties of RNA-targeting ligands, leading us to propose the existence of an "RNA-privileged" chemical space. Biennial updates of the database and the establishment of a website platform (rbind.chem.duke.edu) have provided new insights and tools to design small molecules based on the analyzed physicochemical and spatial properties. In this report and R-BIND 2.0 update, we refined the curation approach and ligand classification system as well as conducted analyses of RNA structure elements for the first time to identify new targeting strategies. Specifically, we curated and analyzed RNA target structural motifs to determine the properties of small molecules that may confer selectivity for distinct RNA secondary and tertiary structures. Additionally, we collected sequences of target structures and incorporated an RNA structure search algorithm into the website that outputs small molecules targeting similar motifs without a priori secondary structure knowledge. Cheminformatic analyses revealed that, despite the 50% increase in small molecule library size, the distinguishing properties of R-BIND ligands remained significantly different from that of proteins and are therefore still relevant to RNA-targeted probe discovery. Combined, we expect these novel insights and website features to enable the rational design of RNA-targeted ligands and to serve as a resource and inspiration for a variety of scientists interested in RNA targeting.
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ARN , Bibliotecas de Moléculas Pequeñas , Bases de Datos de Ácidos Nucleicos , Ligandos , ARN/metabolismo , Sondas ARN , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, emphasized the urgent need for development of novel antivirals. Small-molecule chemical probes offer both to reveal aspects of virus replication and to serve as leads for antiviral therapeutic development. Here, we report on the identification of amiloride-based small molecules that potently inhibit OC43 and SARS-CoV-2 replication through targeting of conserved structured elements within the viral 5'-end. Nuclear magnetic resonancebased structural studies revealed specific amiloride interactions with stem loops containing bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Amilorides represent the first antiviral small molecules that target RNA structures within the 5' untranslated regions and proximal region of the CoV genomes. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNAtargeted antivirals.
RESUMEN
7SK small nuclear RNA (snRNA) is an abundant and ubiquitously expressed noncoding RNA that functions to modulate the activity of RNA Polymerase II (RNAPII) in part by stabilizing distinct pools of 7SK-protein complexes. Prevailing models suggest that the secondary structure of 7SK is dynamically remodeled within its alternative RNA-protein pools such that its architecture differentially regulates the exchange of cognate binding partners. The nuclear hnRNP A1/A2 proteins influence the biology of 7SK snRNA via processes that require an intact stem loop (SL) 3 domain; however, the molecular details by which hnRNPs assemble onto 7SK snRNA are yet to be described. Here, we have taken an integrated approach to present a detailed description of the 7SK-hnRNP A1 complex. We show that unbound 7SK snRNA adopts at least two major conformations in solution, with significant structural differences localizing to the SL2-3 linker and the base of SL3. Phylogenetic analysis indicates that this same region is the least genetically conserved feature of 7SK snRNA. By performing DMS modifications with the presence of excess protein, we reveal that hnRNP A1 binds with selectivity to SL3 through mechanisms that increase the flexibility of the RNA adjacent to putative binding sites. Calorimetric titrations further validate that hnRNP A1-SL3 assembly is complex with the affinity of discrete binding events modulated by the surrounding RNA structure. To interpret this context-dependent binding phenomenon, we determined a 3D model of SL3 to show that it folds to position minimal hnRNP A1/A2 binding sites (5'-Y/RAG-3') within different local environments. SL3-protein complexes resolved by SEC-MALS-SAXS confirm that up to four hnRNP A1 proteins bind along the entire surface of SL3 via interactions that preserve the overall structural integrity of this domain. In sum, the collective results presented here reveal a specific role for a folded SL3 domain to scaffold hnRNP A1/A2-7SK assembly via mechanisms modulated by the surrounding RNA structure.
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Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Conformación de Ácido Nucleico , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Factor B de Elongación Transcripcional Positiva/metabolismo , Unión Proteica , Especificidad por SustratoRESUMEN
The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.
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COVID-19/prevención & control , Espectroscopía de Resonancia Magnética/métodos , Conformación de Ácido Nucleico , ARN Viral/química , SARS-CoV-2/genética , Regiones no Traducidas 3'/genética , Secuencia de Bases , COVID-19/epidemiología , COVID-19/virología , Sistema de Lectura Ribosómico/genética , Genoma Viral/genética , Humanos , Modelos Moleculares , Pandemias , SARS-CoV-2/fisiologíaRESUMEN
Enterovirus 71 (EV71) poses serious threats to human health, particularly in Southeast Asia, and no drugs or vaccines are available. Previous work identified the stem loop II structure of the EV71 internal ribosomal entry site as vital to viral translation and a potential target. After screening an RNA-biased library using a peptide-displacement assay, we identify DMA-135 as a dose-dependent inhibitor of viral translation and replication with no significant toxicity in cell-based studies. Structural, biophysical, and biochemical characterization support an allosteric mechanism in which DMA-135 induces a conformational change in the RNA structure that stabilizes a ternary complex with the AUF1 protein, thus repressing translation. This mechanism is supported by pull-down experiments in cell culture. These detailed studies establish enterovirus RNA structures as promising drug targets while revealing an approach and mechanism of action that should be broadly applicable to functional RNA targeting.
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Enterovirus Humano A/genética , Enterovirus Humano A/fisiología , Infecciones por Enterovirus/metabolismo , Interacciones Huésped-Patógeno/fisiología , Sitios Internos de Entrada al Ribosoma/fisiología , Replicación Viral/fisiología , Regiones no Traducidas 5' , Línea Celular , Infecciones por Enterovirus/virología , Regulación Viral de la Expresión Génica , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , ARN Viral/química , Proteínas Virales/metabolismoRESUMEN
The analysis of protein-nucleic acid interactions is essential for biophysics related research. However, simple, rapid, and accurate methods for quantitative analysis of biomolecular interactions are lacking. We herein establish an electrochemical biosensor approach for protein-nucleic acid binding analysis. Nanoparticle based sensors are fabricated by highly-controlled inkjet printing followed by plasma conversion. A novel bioconjugation method is demonstrated as a simple and rapid approach for protein-based biosensor fabrication. As a proof of concept, we analyzed the binding interaction between unwinding protein 1 (UP1) and SL3ESS3 RNA, confirming the accuracy of this nanoparticle based electrochemical biosensor approach with traditional biophysical methods. We further accurately profiled and differentiated a unique binding interaction pattern of multiple G-tract nucleic acid sequences with heterogeneous nuclear ribonucleoprotein H1. Our study provides insights into a potentially universal platform for in vitro biomolecule interaction analysis using a nanoparticle based electrochemical biosensor approach.
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Técnicas Biosensibles , ADN/química , Oro/química , Ribonucleoproteínas Nucleares Heterogéneas/química , Nanopartículas del Metal/química , ARN Lider Empalmado/química , Técnicas ElectroquímicasRESUMEN
A noninvasive, highly sensitive universal immunosensor platform for protein-based biomarker detection is described in this Article. A neutral charged sensing environment is constructed by an antibody with an oppositely charged amino acid as surface charge neutralizer. By adjusting the pH condition of the testing environment, this neutral charged immunosensor (NCI) directly utilizes the electrostatic charges of the analyte for quantification of circulating protein markers, achieving a wide dynamic range covering through the whole picomole level. Comparing with previous studies on electrostatic charges characterization, this NCI demonstrates its capability to analyze not only the negatively charged biomolecules but also positively charged analytes. We applied this NCI for the detection of HE4 antigen with a detection limit at 2.5 pM and Tau antigen with a detection limit at 0.968 pM, demonstrating the high-sensitivity property of this platform. Furthermore, this NCI possesses a simple fabrication method (less than 2 h) and a short testing turnaround time (less than 30 min), providing an excellent potential for further clinical point-of-care applications.
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Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAP/análisis , Proteínas tau/sangre , Anticuerpos Inmovilizados/inmunología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/inmunología , Complejos de Coordinación/química , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Oro/química , Humanos , Concentración de Iones de Hidrógeno , Inmunoensayo/instrumentación , Inmunoensayo/métodos , Límite de Detección , Oxidación-Reducción , Péptidos/química , Ácido Poliglutámico/química , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAP/inmunología , Proteínas tau/inmunologíaRESUMEN
Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) F/H family are multipurpose RNA binding proteins that participate in most stages of RNA metabolism. Despite having similar RNA sequence preferences, hnRNP F/H proteins function in overlapping and, in some cases, distinct cellular processes. The domain organization of hnRNP F/H proteins is modular, consisting of N-terminal tandem quasi-RNA recognition motifs (F/HqRRM1,2) and a third C-terminal qRRM3 embedded between glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker, with several amino acids strictly conserved between hnRNP H and F. A significant difference occurs at position 105 of the linker, where hnRNP H contains a proline and hnRNP F an alanine. To investigate the influence of P105 on the conformational properties of hnRNP H, we probed the structural dynamics of its HqRRM1,2 domain with X-ray crystallography, NMR spectroscopy, and small-angle X-ray scattering. The collective results best describe that HqRRM1,2 exists in a conformational equilibrium between compact and extended structures. The compact structure displays an electropositive surface formed at the qRRM1-qRRM2 interface. Comparison of NMR relaxation parameters, including Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, between HqRRM1,2 and FqRRM1,2 indicates that FqRRM1,2 primarily adopts a more extended and flexible conformation. Introducing the P105A mutation into HqRRM1,2 alters its conformational dynamics to favor an extended structure. Thus, our work demonstrates that the linker compositions confer different structural properties between hnRNP F/H family members that might contribute to their functional diversity.
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Ribonucleoproteínas Nucleares Heterogéneas/química , Motivo de Reconocimiento de ARN , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/aislamiento & purificación , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
A simple-prepare, single-use and cost-effective, in vitro biosensor for the detection of TAR DNA-binding protein 43 (TDP-43), a biomarker of neuro-degenerative disorders, was designed, manufactured and tested. This study reports the first biosensor application for the detection of TDP-43 using a novel biosensor fabrication methodology. Bioconjugation mechanism was applied by conjugating anti-TDP 43 with N-succinimidyl S-acetylthioacetate (SATA) producing a thiol-linked anti-TDP 43, which was used to directly link with gold electrode surface, minimizing the preparation steps for biosensor fabrication and simplifying the biosensor surface. The effectiveness of this bioconjugation mechanism was evaluated and confirmed by FqRRM12 protein, using nuclear magnetic resonance (NMR). The surface coverage of the electrode was analyzed by Time-of-Flight-Secondary Ion Mass Spectrometry (TOF-SIMS). Differential pulse voltammetry (DPV) was acted as the detection transduction mechanism with the use of [Fe(CN)6]3-/4-redox probe. Human TDP-43 peptide of 0.0005⯵g/mL to 2⯵g/mL in undiluted human serum was analyzed using this TDP-43 biosensor. Interference study of the TDP-43 biosensor using ß-amyloid 42 protein and T-tau protein confirmed the specificity of this TDP-43 biosensor. This bioconjugation chemistry based approach for biosensor fabrication circumvents tedious gold surface modification and functionalization while enabling specific detection of TDP-43 in less than 1â¯h with a low fabrication cost of a single biosensor less than $3.
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Técnicas Biosensibles/métodos , Análisis Químico de la Sangre/métodos , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/sangre , Electrodos , Oro/química , Humanos , Enfermedades Neurodegenerativas/sangre , Enfermedades Neurodegenerativas/diagnóstico , Reproducibilidad de los ResultadosRESUMEN
CXCL4 chemokines have antiangiogenic properties, mediated by different mechanisms, including CXCR3 receptor activation. Chemokines have distinct oligomerization states that are correlated with their biological functions. CXCL4 exists as a stable tetramer under physiological conditions. It is unclear whether the oligomerization state impacts CXCL4-receptor interaction. We found that the CXCL4 tetramer is sensitive to pH and salt concentration. Residues Glu28 and Lys50 were important for tetramer formation, and the first ß-strand and the C-terminal helix are critical for dimerization. By mutating the critical residues responsible for oligomerization, we generated CXCL4 mutants that behave as dimers or monomers under neutral/physiological conditions. The CXCL4 monomer acts as the minimal active unit for interacting CXCR3A, and sulfation of N-terminal tyrosine residues on the receptor is important for binding. Noticeably, CXCL4L1, a CXCL4 variant that differs by three residues in the C-terminal helix, could activate CXCR3A. CXCL4L1 showed a higher tendency to dissociate into monomers, but native CXCL4 did not. This result indicates that monomeric CXCL4 behaves like CXCL4L1. Thus, in this chemokine family, being in the monomeric state seems critical for interaction with CXCR3A.
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Factor Plaquetario 4/metabolismo , Receptores CXCR3/metabolismo , Línea Celular , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Factor Plaquetario 4/química , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Receptores CXCR3/químicaRESUMEN
Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well.
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Factor 2 de Crecimiento de Fibroblastos/aislamiento & purificación , Heparina/química , Multimerización de Proteína , Sitios de Unión , Línea Celular , Cromatografía de Afinidad , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
Electrostatic interaction is a major driving force in the binding of proteins to highly acidic glycosaminoglycan, such as heparin. Although NMR backbone chemical shifts have generally been used to identify the heparin-binding site on a protein, however, there is no correlation between the binding free energies and the perturbed backbone chemical shifts for individual residues. The binding event occurs at the end of a side chain of basic residue, and does not require causing significant alterations in the backbone environment at a distance of multiple bonds. We used the H2CN NMR pulse sequence to detect heparin binding through the side-chain resonances Hε-Cε-Nζ of Lys and Hδ-Cδ-Nε of Arg in the two proteins of hepatoma-derived growth factor (HDGF) and basic fibroblast growth factor (FGF2). H2CN titration experiments revealed chemical shift perturbations in the side chains, which were correlated with the free energy changes in various mutants. The residues K19 in HDGF and K125 in FGF2 demonstrated the most significant perturbations, consistent with our previous observation that the two residues are crucial for binding. The result suggests that H2CN NMR provides a precise evaluation for the electrostatic interactions. The discrepancy observed between backbone and side chain chemical shifts is correlated to the solvent accessibility of residues that the K19 and K125 backbones are highly buried with the restricted backbone conformation and are not strongly affected by the events at the end of the side chains.