RESUMEN
The 10-23 DNAzyme is one of the most prominent catalytically active DNA sequences1,2. Its ability to cleave a wide range of RNA targets with high selectivity entails a substantial therapeutic and biotechnological potential2. However, the high expectations have not yet been met, a fact that coincides with the lack of high-resolution and time-resolved information about its mode of action3. Here we provide high-resolution NMR characterization of all apparent states of the prototypic 10-23 DNAzyme and present a comprehensive survey of the kinetics and dynamics of its catalytic function. The determined structure and identified metal-ion-binding sites of the precatalytic DNAzyme-RNA complex reveal that the basis of the DNA-mediated catalysis is an interplay among three factors: an unexpected, yet exciting molecular architecture; distinct conformational plasticity; and dynamic modulation by metal ions. We further identify previously hidden rate-limiting transient intermediate states in the DNA-mediated catalytic process via real-time NMR measurements. Using a rationally selected single-atom replacement, we could considerably enhance the performance of the DNAzyme, demonstrating that the acquired knowledge of the molecular structure, its plasticity and the occurrence of long-lived intermediate states constitutes a valuable starting point for the rational design of next-generation DNAzymes.
Asunto(s)
Biocatálisis , ADN Catalítico/química , ADN Catalítico/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , ARN/metabolismo , Cinética , Metales/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Factores de TiempoRESUMEN
Membrane proteins can be examined in near-native lipid-bilayer environments with the advent of polymer-encapsulated nanodiscs. These nanodiscs self-assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that have previously been restricted to soluble or detergent-solubilized proteins. Often, however, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques. Thus, the authors aim to fabricate electroneutral-yet water-soluble-polymer nanodiscs. By attaching a sulfobetaine group to the commercial polymers DIBMA and SMA(2:1), these polyanionic polymers are converted to the electroneutral maleimide derivatives, Sulfo-DIBMA and Sulfo-SMA(2:1). Sulfo-DIBMA and Sulfo-SMA(2:1) readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutral nanodiscs avert unspecific interactions, thereby enabling new insights into protein-lipid interactions through lab-on-a-chip detection and in vitro translation of membrane proteins. Finally, the authors create a library comprising thousands of human membrane proteins and use proteome profiling by mass spectrometry to show that protein complexes are preserved in electroneutral nanodiscs.
Asunto(s)
Membrana Dobles de Lípidos , Nanoestructuras , Humanos , Membrana Dobles de Lípidos/química , Polímeros/química , Maleatos/química , Proteínas de la Membrana/química , Nanoestructuras/químicaRESUMEN
Therapeutically relevant proteins such as GPCRs, antibodies and kinases face clear limitations in NMR studies due to the challenges in site-specific isotope labeling and deuteration in eukaryotic expression systems. Here we describe an efficient and simple method to observe the methyl groups of leucine residues in proteins expressed in bacterial, eukaryotic or cell-free expression systems without modification of the expression protocol. The method relies on simple stereo-selective 13 C-labeling and deuteration of leucine that alleviates the need for additional deuteration of the protein. The spectroscopic benefits of "local" deuteration are examined in detail through Forbidden Coherence Transfer (FCT) experiments and simulations. The utility of this labeling method is demonstrated in the cell-free synthesis of bacteriorhodopsin and in the insect-cell expression of the RRM2 domain of human RBM39.
Asunto(s)
Eucariontes/química , Resonancia Magnética Nuclear Biomolecular , Receptores Acoplados a Proteínas G/química , Humanos , Estructura MolecularRESUMEN
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR spectroscopy, and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes.
Asunto(s)
Ácidos Nucleicos/química , Ribonucleoproteína Nuclear Pequeña U1/química , Cristalización , Espectroscopía de Resonancia Magnética , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Deoxyribozymes (DNAzymes) are single-stranded DNA molecules that catalyze a broad range of chemical reactions. The 10-23 DNAzyme catalyzes the cleavage of RNA strands and can be designed to cleave essentially any target RNA, which makes it particularly interesting for therapeutic and biosensing applications. The activity of this DNAzyme in vitro is considerably higher than in cells, which was suggested to be a result of the low intracellular concentration of bioavailable divalent cations. While the interaction of the 10-23 DNAzyme with divalent metal ions was studied extensively, the influence of monovalent metal ions on its activity remains poorly understood. Here, we characterize the influence of monovalent and divalent cations on the 10-23 DNAzyme utilizing functional and biophysical techniques. Our results show that Na+ and K+ affect the binding of divalent metal ions to the DNAzyme:RNA complex and considerably modulate the reaction rates of RNA cleavage. We observe an opposite effect of high levels of Na+ and K+ concentrations on Mg2+- and Mn2+-induced reactions, revealing a different interplay of these metals in catalysis. Based on these findings, we propose a model for the interaction of metal ions with the DNAzyme:RNA complex.
Asunto(s)
ADN Catalítico/metabolismo , ADN de Cadena Simple/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Sitios de Unión , Biocatálisis , ADN Catalítico/química , ADN de Cadena Simple/química , Iones/química , Iones/metabolismo , Potasio/química , Sodio/químicaRESUMEN
Deoxyribozymes (DNAzymes) with RNA hydrolysis activity have a tremendous potential as gene suppression agents for therapeutic applications. The most extensively studied representative is the 10-23 DNAzyme consisting of a catalytic loop and two substrate binding arms that can be designed to bind and cleave the RNA sequence of interest. The RNA substrate is cleaved between central purine and pyrimidine nucleotides. The activity of this DNAzyme in vitro is considerably higher than in vivo, which was suggested to be related to its divalent cation dependency. Understanding the mechanism of DNAzyme catalysis is hindered by the absence of structural information. Numerous biological studies, however, provide comprehensive insights into the role of particular deoxynucleotides and functional groups in DNAzymes. Here we provide an overview of the thermodynamic properties, the impact of nucleobase modifications within the catalytic loop, and the role of different metal ions in catalysis. We point out features that will be helpful in developing novel strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. Consideration of these features will enable to develop improved strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. These insights provide the basis for improving activity in cells and pave the way for developing DNAzyme applications.
Asunto(s)
ADN Catalítico/química , ADN de Cadena Simple/química , Metales/química , Conformación de Ácido Nucleico , Cationes BivalentesRESUMEN
Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria and active in the nanomolar range. Nisin is the most intensely studied and used lantibiotic, with applications as food preservative and recognized potential for clinical usage. However, different bacteria that are pathogenic for humans and do not produce nisin, including Streptococcus agalactiae, show an innate resistance that has been related to the nisin resistance protein (NSR), a membrane-associated protease. Here, we report the first-in-class small-molecule inhibitors of SaNSR identified by virtual screening based on a previously derived structural model of the nisin/NSR complex. The inhibitors belong to three different chemotypes, of which the halogenated phenyl-urea derivative NPG9 is the most potent one. Co-administration of NPG9 with nisin yields increased potency compared to nisin alone in SaNSR-expressing bacteria. The binding mode of NPG9, predicted with molecular docking and validated by extensive molecular dynamics simulations, confirms a structure-activity relationship derived from the in vivo data. Saturation transfer difference-NMR experiments demonstrate direct binding of NPG9 to SaNSR and agree with the predicted binding mode. Our results demonstrate the potential to overcome SaNSR-related lantibiotic resistance by small molecules.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Farmacorresistencia Bacteriana/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Streptococcus agalactiae/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Streptococcus agalactiae/química , Relación Estructura-ActividadRESUMEN
Intrinsically disordered proteins dynamically sample a wide conformational space and therefore do not adopt a stable and defined three-dimensional conformation. The structural heterogeneity is related to their proper functioning in physiological processes. Knowledge of the conformational ensemble is crucial for a complete comprehension of this kind of proteins. We here present an approach that utilizes dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of sparsely isotope-labeled proteins in frozen solution to take snapshots of the complete structural ensembles by exploiting the inhomogeneously broadened line-shapes. We investigated the intrinsically disordered protein α-synuclein (α-syn), which plays a key role in the etiology of Parkinson's disease, in three different physiologically relevant states. For the free monomer in frozen solution we could see that the so-called "random coil conformation" consists of α-helical and ß-sheet-like conformations, and that secondary chemical shifts of neighboring amino acids tend to be correlated, indicative of frequent formation of secondary structure elements. Based on these results, we could estimate the number of disordered regions in fibrillar α-syn as well as in α-syn bound to membranes in different protein-to-lipid ratios. Our approach thus provides quantitative information on the propensity to sample transient secondary structures in different functional states. Molecular dynamics simulations rationalize the results.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , alfa-Sinucleína/química , Secuencia de Aminoácidos , Simulación de Dinámica Molecular , Conformación Proteica , TemperaturaRESUMEN
Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4-mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4-mediated phosphoproteome, including construction of kinase-substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, δ-catenin, and PPP1Cα in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy.
Asunto(s)
Neoplasias de la Mama/genética , Quimiocina CXCL12/metabolismo , Retroalimentación Fisiológica/fisiología , Metástasis de la Neoplasia/fisiopatología , Células Madre Neoplásicas/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal/genética , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones SCID , Fosforilación , Proteómica/métodosRESUMEN
Membrane proteins act as a central interface between the extracellular environment and the intracellular response and as such represent one of the most important classes of drug targets. The characterization of the molecular properties of integral membrane proteins, such as topology and interdomain interaction, is key to a fundamental understanding of their function. Atomic force microscopy (AFM) and force spectroscopy have the intrinsic capabilities of investigating these properties in a near-native setting. However, atomic force spectroscopy of membrane proteins is traditionally carried out in a crystalline setup. Alternatively, model membrane systems, such as tethered bilayer membranes, have been developed for surface-dependent techniques. While these setups can provide a more native environment, data analysis may be complicated by the normally found statistical orientation of the reconstituted protein in the model membrane. We have developed a model membrane system that enables the study of membrane proteins in a defined orientation by single-molecule force spectroscopy. Our approach is demonstrated using cell-free expressed bacteriorhodopsin coupled to a quartz glass surface in a defined orientation through a protein anchor and reconstituted inside an artificial membrane system. This approach offers an effective way to study membrane proteins in a planar lipid bilayer. It can be easily transferred to all membrane proteins that possess a suitable tag and can be reconstituted into a lipid bilayer. In this respect, we anticipate that this technique may contribute important information on structure, topology, and intra- and intermolecular interactions of other seven-transmembrane helical receptors.
Asunto(s)
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Membranas Artificiales , Microscopía de Fuerza Atómica , Propiedades de SuperficieRESUMEN
A growing number of nuclear magnetic resonance (NMR) spectroscopic studies are impaired by the limited information content provided by the standard set of experiments conventionally recorded. This is particularly true for studies of challenging biological systems including large, unstructured, membrane-embedded and/or paramagnetic proteins. Here we introduce the concept of unified time-optimized interleaved acquisition NMR (UTOPIA-NMR) for the unified acquisition of standard high-γ (e.g. (1)H) and low-γ (e.g. (13)C) detected experiments using a single receiver. Our aim is to activate the high level of polarization and information content distributed on low-γ nuclei without disturbing conventional magnetization transfer pathways. We show that using UTOPIA-NMR we are able to recover nearly all of the normally non-used magnetization without disturbing the standard experiments. In other words, additional spectra, that can significantly increase the NMR insights, are obtained for free. While we anticipate a broad range of possible applications we demonstrate for the soluble protein Bcl-xL (ca. 21 kDa) and for OmpX in nanodiscs (ca. 160 kDa) that UTOPIA-NMR is particularly useful for challenging protein systems including perdeuterated (membrane) proteins.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas de la Membrana/químicaRESUMEN
The choice of a suitable membrane mimicking environment is of fundamental importance for the characterization of structure and function of membrane proteins. In this respect, usage of the lipid bilayer nanodisc technology provides a unique potential for nuclear magnetic resonance (NMR)-based studies. This review summarizes the recent advances in this field, focusing on (i) the strengths of the system, (ii) the bottlenecks that may be faced, and (iii) promising capabilities that may be explored in future studies.
Asunto(s)
Membrana Dobles de Lípidos/química , Espectroscopía de Resonancia Magnética/métodos , Nanoestructuras/química , Nanotecnología/métodos , Animales , Humanos , Membrana Dobles de Lípidos/metabolismoRESUMEN
Nuclear magnetic resonance (NMR) spectroscopy has the intrinsic capabilities to investigate proteins in native environments. In general, however, NMR relies on non-natural protein purity and concentration to increase the desired signal over the background. We here report on the efficient and specific hyperpolarization of low amounts of a target protein in a large isotope-labeled background by combining dynamic nuclear polarization (DNP) and the selectivity of protein interactions. Using a biradical-labeled ligand, we were able to direct the hyperpolarization to the protein of interest, maintaining comparable signal enhancement with about 400-fold less radicals than conventionally used. We could selectively filter out our target protein directly from crude cell lysate obtained from only 8â mL of fully isotope-enriched cell culture. Our approach offers effective means to study proteins with atomic resolution in increasingly native concentrations and environments.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Isótopos de Carbono/química , Óxidos N-Cíclicos/química , Marcaje Isotópico , Polietilenglicoles/química , Propanoles/química , Estructura Secundaria de Proteína , Proteínas/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/química , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína bcl-X/química , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded ß-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/ultraestructura , Bacteriorodopsinas/química , Bacteriorodopsinas/ultraestructura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , Hidrolasas/química , Hidrolasas/ultraestructura , Polímeros/química , Propilaminas/química , Solventes/química , Tensoactivos/química , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas/métodos , Estructura Terciaria de Proteína , SolubilidadRESUMEN
While amphipols have been proven useful for refolding of seven transmembrane helical (7-TM) proteins including G-protein-coupled receptors (GPCRs) and it could be shown that an amphipol environment is in principle suitable for NMR structural studies of the embedded protein, high-resolution NMR insights into amphipol refolded and isotopically labeled GPCRs are still very limited. Here we report on the recent progress toward NMR structural studies of the melanocortin-2 and -4 receptors, two class A GPCRs which so far have not been reported to be incorporated into an amphipol environment. Making use of the established 7-TM protein bacteriorhodopsin (BR) we initially tested and optimized amphipol refolding conditions. Most promising conditions were transferred to the refolding of the two melanocortin receptors. Analytical-scale refolding experiments on the melanocortin-2 receptor show very similar behavior to the results obtained on BR. Using cell-free protein expression we could generate sufficient amounts of isotopically labeled bacteriorhodopsin as well as melanocortin-2 and -4 receptors for an initial NMR analysis. Upscaling of the amphipol refolding protocol to protein amounts needed for NMR structural studies was, however, not straightforward and impeded detailed NMR insights for the two GPCRs. While well-resolved and dispersed NMR spectra could only be obtained for bacteriorhodopsin, a comparison of NMR data recorded on the melanocortin-4 receptor in SDS and in an amphipol environment indicates that amphipol refolding induces larger structural modifications in the receptor.
Asunto(s)
Algoritmos , Cromatografía en Gel/métodos , Espectroscopía de Resonancia Magnética/métodos , Polímeros/química , Propilaminas/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/ultraestructura , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
Disc-like lipid nanoparticles stabilized by saponin biosurfactants display fascinating properties, including their temperature-driven re-organization. ß-Aescin, a saponin from seed extract of the horse chestnut tree, shows strong interactions with lipid membranes and has gained interest due to its beneficial therapeutic implications as well as its ability to decompose continuous lipid membranes into size-tuneable discoidal nanoparticles. Here, we characterize lipid nanoparticles formed by aescin and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. We present site-resolved insights into central molecular interactions and their modulations by temperature and aescin content. Using the membrane protein bacteriorhodopsin, we additionally demonstrate that, under defined conditions, aescin-lipid discs can accommodate medium-sized transmembrane proteins. Our data reveal the general capability of this fascinating system to generate size-tuneable aescin-lipid-protein particles, opening the road for further applications in biochemical, biophysical and structural studies.
Asunto(s)
Escina , Nanopartículas , Tamaño de la Partícula , Nanopartículas/química , Escina/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Temperatura , Dimiristoilfosfatidilcolina/química , Estabilidad Proteica , LiposomasRESUMEN
Structural studies of membrane proteins are still hampered by difficulties of finding appropriate membrane-mimicking media that maintain protein structure and function. Phospholipid nanodiscs seem promising to overcome the intrinsic problems of detergent-containing environments. While nanodiscs can offer a near-native environment, the large particle size complicates their routine use in the structural analysis of membrane proteins by solution NMR. Here, we introduce nanodiscs assembled from shorter ApoA-I protein variants that are of markedly smaller diameter and show that the resulting discs provide critical improvements for the structure determination of membrane proteins by NMR. Using the bacterial outer-membrane protein OmpX as an example, we demonstrate that the combination of small nanodisc size, high deuteration levels of protein and lipids, and the use of advanced non-uniform NMR sampling methods enable the NMR resonance assignment as well as the high-resolution structure determination of polytopic membrane proteins in a detergent-free, near-native lipid bilayer setting. By applying this method to bacteriorhodopsin, we show that our smaller nanodiscs can also be beneficial for the structural characterization of the important class of seven-transmembrane helical proteins. Our set of engineered nanodiscs of subsequently smaller diameters can be used to screen for optimal NMR spectral quality for small to medium-sized membrane proteins while still providing a functional environment. In addition to their key improvements for de novo structure determination, due to their smaller size these nanodiscs enable the investigation of interactions between membrane proteins and their (soluble) partner proteins, unbiased by the presence of detergents that might disrupt biologically relevant interactions.
Asunto(s)
Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Nanoestructuras/química , Fosfolípidos/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación ProteicaRESUMEN
Solid-state NMR spectroscopy proved to be a versatile tool for characterization of structure and dynamics of complex biochemical systems. In particular, magic angle spinning (MAS) solid-state NMR came to maturity for application towards structural elucidation of biological macromolecules. Current challenges in applying solid-state NMR as well as progress achieved recently will be discussed in the following chapter focusing on conceptual aspects important for structural elucidation of proteins.
Asunto(s)
Amiloide/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas de la Membrana/metabolismo , Proteínas/química , Anisotropía , Difusión , Diseño de Equipo , Humanos , Espectroscopía de Resonancia Magnética/instrumentación , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Saccharomyces cerevisiae/metabolismoRESUMEN
Understanding the molecular features of catalytically active DNA sequences, so-called DNAzymes, is essential not only for our understanding of the fundamental properties of catalytic nucleic acids in general, but may well be the key to unravelling their full potential via tailored modifications. Our recent findings contributed to the endeavour to assemble a mechanistic picture of DNA-mediated catalysis by providing high-resolution structural insights into the 10-23 DNAzyme (Dz) and exposing a complex interplay between the Dz's unique molecular architecture, conformational plasticity, and dynamic modulation by metal ions as central elements of the DNA catalyst. Here, we discuss key features of our findings and compare them to other studies on similar systems.
Asunto(s)
ADN Catalítico , ADN Catalítico/genética , ADN Catalítico/química , ADN Catalítico/metabolismo , ADN/genética , ADN/química , Metales/química , Secuencia de Bases , CatálisisRESUMEN
Catalytically active DNA oligomers (or DNAzymes) offer a broad spectrum of functions as well as applications. Although known for over two decades, the DNAzyme's mode-of-actions are still poorly understood, mainly due to lack of high-resolution structural insights. Due to their molecular size, structural flexibility, and dynamic interactions with metal-ion cofactors, solution nuclear magnetic resonance spectroscopy (NMR) can serve as optimal tool to obtain mechanistic insights of DNAzymes. In this respect, nearly all states of the DNAzyme and its substrate during the catalytic cycle are accessible. The instructions and protocols provided in the following may assist the initial steps of an NMR-based characterization of DNAzymes. To reduce the initial setup requirements and foster exciting new research projects, the discussed approaches focus on experiments that do not require cost-intensive isotope labeling strategies.