RESUMO
RNA nanotechnology uses RNA structural motifs to build nanosized architectures that assemble through selective base-pair interactions. Herein, we report the crystal-structure-guided design of highly stable RNA nanotriangles that self-assemble cooperatively from short oligonucleotides. The crystal structure of an 81 nucleotide nanotriangle determined at 2.6â Å resolution reveals the so-far smallest circularly closed nanoobject made entirely of double-stranded RNA. The assembly of the nanotriangle architecture involved RNA corner motifs that were derived from ligand-responsive RNA switches, which offer the opportunity to control self-assembly and dissociation.
Assuntos
Nanoestruturas , Conformação de Ácido Nucleico , RNA/química , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida NativaRESUMO
The internal ribosome entry site (IRES) in the 5' untranslated region (UTR) of the hepatitis C virus (HCV) RNA genome is responsible for initiation of viral protein synthesis. The IRES RNA contains autonomously folding domains that are potential targets for antiviral translation inhibitors. Here, we describe the experimental crystal structure determination of the IRES subdomain IIa in complex with a previously discovered benzimidazole translation inhibitor. The structure of an inhibitor complex of the highly conserved IRES subdomain IIa holds promise for structure-based design of new anti-HCV drugs.
Assuntos
Hepacivirus/química , Sítios Internos de Entrada Ribossomal , Regiões 5' não Traduzidas , Antivirais/química , Sequência de Bases , Benzimidazóis/química , Sítios de Ligação , Cristalografia por Raios X , Genoma Viral , Hepacivirus/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Biossíntese de Proteínas/efeitos dos fármacos , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/química , RNA Viral/genética , Ribossomos/metabolismoRESUMO
Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2'-deoxyuridine-5'-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix-loop-helix domain on the protein surface was identified as the putative RNA-binding site.
Assuntos
Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , RNA Mensageiro/química , Timidilato Sintase/química , Sequência de Bases , Sítios de Ligação/genética , Códon de Iniciação/genética , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Humanos , Espectrometria de Massas/métodos , Modelos Moleculares , Mutagênese , Motivos de Nucleotídeos/genética , Ligação Proteica/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Raios UltravioletaRESUMO
An internal ribosome entry site (IRES) initiates protein synthesis in RNA viruses, including the hepatitis C virus (HCV). We have discovered ligand-responsive conformational switches in viral IRES elements. Modular RNA motifs of greatly distinct sequence and local secondary structure have been found to serve as functionally conserved switches involved in viral IRES-driven translation and may be captured by identical cognate ligands. The RNA motifs described here constitute a new paradigm for ligand-captured switches that differ from metabolite-sensing riboswitches with regard to their small size, as well as the intrinsic stability and structural definition of the constitutive conformational states. These viral RNA modules represent the simplest form of ligand-responsive mechanical switches in nucleic acids.
Assuntos
Hepacivirus/química , Conformação de Ácido Nucleico , RNA Viral/química , Sequências Reguladoras de Ácido Ribonucleico , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/biossínteseRESUMO
2-Aminobenzoxazoles have been synthesized as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The compounds were designed to explore the less basic benzoxazole system as a replacement for the core scaffold in previously discovered benzimidazole viral translation inhibitors. Structure-activity relationships in the target binding of substituted benzoxazole ligands were investigated.
Assuntos
Antivirais/farmacologia , Benzoxazóis/farmacologia , Hepacivirus/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Ribossomos/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , Benzoxazóis/síntese química , Benzoxazóis/química , Hepacivirus/química , Ligantes , Modelos Moleculares , Estrutura Molecular , RNA Viral/metabolismo , Ribossomos/metabolismoRESUMO
We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex.
Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , RNA Viral/genética , RNA Viral/metabolismo , Ribossomos/metabolismo , Antivirais/síntese química , Antivirais/química , Benzimidazóis/síntese química , Benzimidazóis/química , Relação Dose-Resposta a Droga , Ligantes , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The internal ribosome entry site (IRES) in the 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome initiates translation of the viral polyprotein precursor. The unique structure and high sequence conservation of the 5' UTR render the IRES RNA a potential target for the development of selective viral translation inhibitors. Here, we provide an overview of approaches to block HCV IRES function by nucleic acid, peptide, and small molecule ligands. Emphasis will be given to the IRES subdomain IIa, which currently is the most advanced target for small molecule inhibitors of HCV translation. The subdomain IIa behaves as an RNA conformational switch. Selective ligands act as translation inhibitors by locking the conformation of the RNA switch. We review synthetic procedures for inhibitors as well as structural and functional studies of the subdomain IIa target and its ligand complexes.
Assuntos
Hepacivirus/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Regiões 5' não Traduzidas , Hepacivirus/genética , Modelos MolecularesRESUMO
The preparation and crystal structure of 8-chloro-1H-pyrrolo[2,3-b]phenazine 5-oxide (1) are described. Compound 1 formed dark purple crystals from deeply colored solution in methanol. Crystal plates were in the triclinic system, P-1 space group with unit cell parameters a = 6.9514(8), b = 9.1568(10), c = 10.2067(11), α = 84.509(2), ß = 82.936(2), γ = 72.357(2) and a cell volume of 613.25(12) A-3. The title compound which contains the first example of the extensively conjugated pyrrolo-phenazine N-oxide system exhibits strong light absorption in the green to cyan wavelength range which disappears upon protonation.
RESUMO
The crystal structure of 8-((dimethylamino)methyl)-1-(3-(dimethylamino)propyl)-1,7,8,9-tetrahydrochromeno[5,6-d]imidazol-2-amine (1), an inhibitor of the hepatitis C virus internal ribosome entry site, is described and compared to the structure of the compound in complex with the viral RNA target. Compound 1 crystallized by pentane vapor diffusion into dichloroethane solution. It crystallized in the monoclinic system, P21/c space group with unit cell parameters a = 15.7950(5) Å, b = 14.0128(4) Å, c = 8.8147(3) Å, ß = 94.357(2)° and a cell volume of 1945.34(11) A-3. Packing interactions in the small molecule crystal lattice correspond to key interactions of the compound with the viral RNA target.
RESUMO
Three-dimensional RNA models fitted into crystallographic density maps exhibit pervasive conformational ambiguities, geometric errors and steric clashes. To address these problems, we present enumerative real-space refinement assisted by electron density under Rosetta (ERRASER), coupled to Python-based hierarchical environment for integrated 'xtallography' (PHENIX) diffraction-based refinement. On 24 data sets, ERRASER automatically corrects the majority of MolProbity-assessed errors, improves the average R(free) factor, resolves functionally important discrepancies in noncanonical structure and refines low-resolution models to better match higher-resolution models.
Assuntos
Biologia Computacional , RNA/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , SoftwareRESUMO
The internal ribosome entry site (IRES) in the hepatitis C virus (HCV) RNA genome is essential for the initiation of viral protein synthesis. IRES domains adopt well-defined folds that are potential targets for antiviral translation inhibitors. We have determined the three-dimensional structure of the IRES subdomain IIa in complex with a benzimidazole translation inhibitor at 2.2 Å resolution. Comparison to the structure of the unbound RNA in conjunction with studies of inhibitor binding to the target in solution demonstrate that the RNA undergoes a dramatic ligand-induced conformational adaptation to form a deep pocket that resembles the substrate binding sites in riboswitches. The presence of a well-defined ligand-binding pocket within the highly conserved IRES subdomain IIa holds promise for the development of unique anti-HCV drugs with a high barrier to resistance.
Assuntos
Hepacivirus/genética , RNA Viral/química , Riboswitch , Benzimidazóis/farmacologia , Transferência Ressonante de Energia de Fluorescência , Ligantes , Modelos Moleculares , Conformação de Ácido Nucleico , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
The three-dimensional structures of noncoding RNA molecules reveal recurring architectural motifs that have been exploited for the design of artificial RNA nanomaterials. Programmed assembly of RNA nanoobjects from autonomously folding tetraloop-receptor complexes as well as junction motifs has been achieved previously through sequence-directed hybridization of complex sets of long oligonucleotides. Due to size and complexity, structural characterization of artificial RNA nanoobjects has been limited to low-resolution microscopy studies. Here we present the design, construction, and crystal structure determination at 2.2 Å of the smallest yet square-shaped nanoobject made entirely of double-stranded RNA. The RNA square is comprised of 100 residues and self-assembles from four copies each of two oligonucleotides of 10 and 15 bases length. Despite the high symmetry on the level of secondary structure, the three-dimensional architecture of the square is asymmetric, with all four corners adopting distinct folding patterns. We demonstrate the programmed self-assembly of RNA squares from complex mixtures of corner units and establish a concept to exploit the RNA square as a combinatorial nanoscale platform.
Assuntos
Conformação de Ácido Nucleico , RNA/química , Cristalografia por Raios X , RNA/genéticaRESUMO
Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand-RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region.
Assuntos
Aminoglicosídeos/química , Antibacterianos/química , Modelos Moleculares , RNA Ribossômico 16S/química , 2-Aminopurina/química , Sítios de Ligação , Cristalografia por Raios X , Higromicina B/química , Ligantes , Conformação de Ácido NucleicoRESUMO
A synthetic procedure toward 1,3-diazepane scaffolds of natural product-like complexity was developed for the construction of RNA-directed ligand libraries. A molecular building block was designed that combines the characteristics of RNA-binding natural products, including a high density of hydrogen bond donors and acceptors around a rigid, nonplanar scaffold with straightforward total-synthetic accessibility that permits extensive control over the chemical space. The synthesis of the 1,3-diazepane scaffold was achieved via an unprecedented cyanamide-induced rearrangement of epoxy-delta-lactams.
Assuntos
Azepinas/química , Cianamida/química , Compostos de Epóxi/química , Lactamas/química , Ligação de Hidrogênio , Conformação Molecular , EstereoisomerismoRESUMO
The internal ribosome entry site (IRES), a highly conserved structured element of the hepatitis C virus (HCV) genomic RNA, is an attractive target for antiviral drugs. Here we show that benzimidazole inhibitors of the HCV replicon act by conformational induction of a widened interhelical angle in the IRES subdomain IIa, which facilitates the undocking of subdomain IIb from the ribosome and ultimately leads to inhibition of IRES-driven translation in HCV-infected cells.
Assuntos
Hepacivirus/metabolismo , Ribossomos/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , Sequência de Bases , Benzimidazóis/farmacologia , Citosina/química , Genoma Viral/efeitos dos fármacos , Genótipo , Hepacivirus/química , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Magnésio/farmacologia , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Replicon/efeitos dos fármacos , Replicon/genética , Ribossomos/genética , Ribossomos/virologia , Transdução de SinaisAssuntos
Hepacivirus/química , Manganês/química , RNA Viral/química , Tungstênio/química , Cristalização , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/química , Conformação Proteica , Estrutura Secundária de Proteína , Soluções/química , Relação Estrutura-AtividadeRESUMO
Shock-induced crystallization of the supercooled ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate, C8H15N2+.PF6-, allows for the first time precise X-ray diffraction analysis directly pertinent to the fluid state. This intermediate-chain-length structure shows features of both short- and long-chain analogs. Two types of interplanar distances between imidazolium rings are observed. The anions are located in channels formed by the imidazolium rings and alkyl chains.
RESUMO
The reaction of solid [RuClCp(PPh(3))(2)] with TeSe(3)(2-) or Se(n)(2-) in DMF leads to the formation of [RuCp(PPh(3))(mu(2)-Se(2))](2) (1). In the structure of this compound the two bridging Se(2) groups lead to a six-membered Ru(2)Se(4) ring in a chair conformation. Attached to each Ru center is a PPh(3) ligand in an equatorial position and a Cp ring in an axial position. The compound is diamagnetic. The compound [Ru(2)Cp(2)(mu(3)-Se(2))(mu(3)-Se)](2) (2) is obtained under similar conditions in the presence of air. This structure comprises a centrosymmetric Ru(4)Se(6) dimer formed from the two bridging Se groups and the two bridging Se(2) groups. Each Ru center is pi-bonded to a Cp ring. The reaction of solid [RuClCp(PPh(3))(2)] with a Te(n)(2-) polytelluride solution in DMF leads to the diamagnetic compound [(RuCp(PPh(3)))(2)(mu(2)-(1,4-eta:3,6-eta)Te(6))] (3). Here the Ru centers are bound to a bridging Te(6) chain at the 1, 4, 3, and 6 positions, leading to a bicyclic Ru(2)Te(6) ring. Each Ru atom is bound to a Cp ring and a PPh(3) group. This dimer possesses a center of symmetry. The structure of 3 is the first example of a bicyclic complex where fusion occurs along a Te-Te bond. If the same reaction is carried out in DMF/CH(2)Cl(2), rather than DMF, then [(RuCp(PPh(3)))(2)(mu(2)-(1,4-eta:3,6-eta)Te(6))].CH(2)Cl(2) (4) is obtained. In the solid state it possesses the same Ru(2)Te(6) structural unit as does 3, but the unit lacks a crystallographically imposed center of symmetry. The electronic structures of 3 and 4 have been analyzed with the use of first principles density functional theory. Bond order analysis indicates that the Te-Te bond where fusion occurs has a shared bonding charge of about (2)/(3) of that found for Te-Te single bonds.
RESUMO
The [Au2(TeSe2)2]2- anion has been prepared from the reaction of [TeSe3]2- with AuCN in DMF in the presence of PEt3 and from the reaction of [TeSe2]2- with AuCN in DMF. Reaction of [TeSe2]2- with AuCN in DMF in the presence of PEt3 leads ultimately to the [Au2(Te2)2]2- anion.