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The design of hybrid nucleic acid nanomaterials capitalizes on the partitioning of architectural and functional roles between structurally diverse RNA modules and chemically robust DNA components. Selecting optimal combinations of RNA and DNA building blocks is the key to preparing stable polygonal RNA-DNA hybrid nanoshapes. Here, we outline a simple screening strategy by gel electrophoresis under native folding conditions to identify combinations of RNA and DNA modules that self-assemble to robust polygonal hybrid nanoshapes. As a proof of concept, we outline the preparation of RNA-DNA hybrid nanoshapes containing a set of different RNA architectural joints, including internal loop motifs and three-way junction (3WJ) folds. For each hybrid nanoshape, we demonstrate the selection process used to identify optimal DNA modules from a library of DNA connectors. The simple screening strategy outlined here provides a general robust method to identify and prepare RNA-DNA hybrid nanoshapes from diverse libraries of discrete nucleic acid building blocks.

Design and preparation of layered composite materials alternating between nucleic acids and proteins has been elusive due to limitations in occurrence and geometry of interaction sites in natural biomolecules. We report the design and kinetically controlled stepwise synthesis of a nano-sandwich composite by programmed noncovalent association of protein, DNA and RNA modules. A homo-tetramer protein core was introduced to control the self-assembly and precise positioning of two RNA–DNA hybrid nanotriangles in a co-parallel sandwich arrangement. Kinetically favored self-assembly of the circularly closed nanostructures at the protein was driven by the intrinsic fast folding ability of RNA corner modules which were added to precursor complex of DNA bound to the protein. The 3D architecture of this first synthetic protein–RNA–DNA complex was confirmed by fluorescence labeling and cryo-electron microscopy studies. The synthesis strategy for the nano-sandwich composite provides a general blueprint for controlled noncovalent assembly of complex supramolecular architectures from protein, DNA and RNA components, which expand the design repertoire for bottom-up preparation of layered biomaterials.

Hybrid nucleic acid nanostructures partition architectural and functional roles between ribonucleic acid (RNA) joints and deoxyribonucleic acid (DNA) connectors. Nanoshapes self-assemble from nucleic acid modules through synergistic stabilization of marginally stable base pairing interactions within circularly closed polygons. Herein, we report the development of hybrid nanoshapes that include multiple different RNA modules such as internal loop and three-way junction (3WJ) motifs. An iterative mix-and-match screening approach was used to identify suitable DNA connectors that furnished stable nanoshapes for combinations of different RNA modules. The resulting complex multicomponent RNA-DNA hybrid nanoshapes were characterized by atomic force microscopy (AFM) imaging. Our research provides proof of concept for modular design, assembly and screening of RNA-DNA hybrid nanoshapes as building blocks for complex extended nucleic acid materials with features at the sub-10 nm scale.

The preparation of nucleic acid nanostructures has relied predominantly on procedures of additive fabrication in which complex architectures are assembled by concerted self-assembly and sequential addition of building blocks. We had previously established RNA-DNA hybrid nanoshapes with modular architectures that enable multistep synthetic approaches inspired by organic molecular synthesis where additive and transformative steps are used to prepare complex molecular architectures. We report the establishment of module replacement and strand exchange as synthetic transformations in nucleic acid hybrid nanoshapes, which are enabled by minimally destabilizing sequence elements such as a single unpaired overhang nucleotide or a mismatch base pair. Module exchange facilitated by thermodynamic lability triggers adds a powerful transformative approach to the repertoire of additive and transformative synthetic methods for the preparation of complex composite materials.

Mass-selective threshold photoelectron spectroscopy in the gas phase was employed to characterize the dialkynyl triplet carbenes pentadiynylidene (HC5H), methylpentadiynylidene (MeC5H), and dimethylpentadiynylidene (MeC5Me). Diazo compounds were employed as precursors to generate the carbenes by flash pyrolysis. The R1-C5-R2 carbon chains were photoionized by vacuum ultraviolet (VUV) synchrotron radiation in photoelectron photoion coincidence (PEPICO) experiments. High-level ab initio computations were carried out to support the interpretation of the experiments. For the unsubstituted pentadiynylidene (R1 = R2 = H) the recorded spectrum yields an adiabatic ionization energy (IEad) of 8.36 ± 0.03 eV. In addition, a second carbene isomer, 3-(didehydrovinylidene)cyclopropene, with a singlet electronic ground state, was identified in the spectrum based on the IEad of 8.60 ± 0.03 eV and Franck-Condon simulations. We found that multireference computations are required to reliably calculate the IEad for this molecule. CASPT2 computations predicted an IEad = 8.55 eV, while coupled-cluster computations significantly overestimate the IE. The cyclic isomer is most likely formed from another isomer of the precursor present in the sample. Stepwise methyl-substitution of the carbene leads to a reduction of the IE to 7.77 ± 0.04 eV for methylpentadiynylidene and 7.27 ± 0.06 eV for dimethylpentadiynylidene. The photoionization and dissociative photoionization of the precursors is investigated as well.

Comprehensive understanding of the structure–function relationship of RNA both in real time and at atomic level will have a profound impact in advancing our understanding of RNA functions in biology. Here, we describe the first example of a multifunctional nucleoside probe, containing a conformation‐sensitive fluorophore and an anomalous X‐ray diffraction label (5‐selenophene uracil), which enables the correlation of RNA conformation and recognition under equilibrium and in 3D. The probe incorporated into the bacterial ribosomal RNA decoding site, fluorescently reports antibiotic binding and provides diffraction information in determining the structure without distorting native RNA fold. Further, by comparing solution binding data and crystal structure, we gained insight on how the probe senses ligand‐induced conformational change in RNA. Taken together, our nucleoside probe represents a new class of biophysical tool that would complement available tools for functional RNA investigations.

Self-assembly of nucleic acid nanostructures is driven by selective association of oligonucleotide modules through base pairing between complementary sequences. Herein, we report the development of RNA–DNA hybrid nanoshapes that conditionally assemble under the control of an adenosine ligand. The design concept for the nanoshapes relies on ligand-dependent stabilization of DNA aptamers that serve as connectors between marginally stable RNA corner modules. Ligand-dependent RNA–DNA nanoshapes self-assemble in an all-or-nothing process by coupling adenosine binding to the formation of circularly closed structures which are stabilized through continuous base stacking in the resulting polygons. By screening combinations of various DNA aptamer constructs with RNA corner modules for the formation of stable complexes, we identified adenosine-dependent nanosquares whose shape was confirmed by atomic force microscopy. As a proof-of-concept for sensor applications, adenosine-responsive FRET-active nanosquares were obtained by dye conjugation of the DNA aptamer components.

DNA and RNA have emerged as a material for nanotechnology applications that take advantage of the nucleic acids’ ability to encode folding and programmable self-assembly through mainly base pairing. The two types of nucleic acid have rarely been used in combination to enhance structural diversity or for partitioning of functional and architectural roles. Here, we report a design and screening strategy to integrate combinations of RNA motifs as architectural joints and DNA building blocks as functional modules for programmable self-assembly of a versatile toolkit of polygonal nucleic acid nanoshapes. Clean incorporation of diverse DNA modules with various topologies attest to the extraordinary robustness of the RNA-DNA hybrid framework. The design and screening strategy enables systematic development of RNA-DNA hybrid nanoshapes as programmable platforms for applications in molecular recognition, sensor and catalyst development as well as protein interaction studies., DNA and RNA have been used for nanotechnology applications, though rarely in combination. Here the authors report the use of RNA motifs as structural joints with DNA building blocks for enhanced construction of small multi-component nanoshapes.

The function of RNA switches involved in the regulation of transcription and translation relies on their ability to adopt different, structurally well-defined states. A new class of ligand-responsive RNA switches, which we recently discovered in positive strand RNA viruses, are distinct from conventional riboswitches. The viral switches undergo large conformational changes in response to ligand binding while retaining the same secondary structure in their free and ligand-bound forms. Here, we describe FRET experiments to study folding and ligand binding of the viral RNA switches. In addition to reviewing previous approaches involving RNA model constructs which were directly conjugated with fluorescent dyes, we outline the design and application of new modular constructs for FRET experiments, in which dye labeling is achieved by hybridization of a core RNA switch module with universal DNA fluorescent probes. As an example, folding and ligand binding of the RNA switch from the internal ribosome entry site of hepatitis C virus is studied comparatively with conventional and modular FRET constructs.

A series of 2-aminobenzimidazole analogues have been synthesised and tested for binding to a previously established RNA target for viral translation inhibitors in the internal ribosome entry site (IRES) of the hepatitis C virus (HCV). Synthesis of new inhibitor compounds followed a highly convergent strategy which allowed for incorporation of diverse tertiary amino substituents in high overall yields (eight-steps, 4–22%). Structure–activity relationship (SAR) studies focussed on the tertiary amine substituent involved in hydrogen bonding with the RNA backbone at the inhibitor binding site. The SAR study was further correlated with in silico docking experiments. Analogous compounds showed promising activities (half maximal effective concentration, EC50: 21–89µM). Structures of the synthesised analogues and a correlation to their mode of binding, provided the opportunity to explore parameters required for selective targeting of the HCV IRES at the subdomain IIa which acts as an RNA conformational switch in HCV translation.

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.

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.

The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay for similarly acting HCV translation inhibitors. The assay relies on monitoring fluorescence changes that indicate rearrangement of the RNA target conformation upon ligand binding. Screening of a small pilot set of potential RNA binders identified a benzoxazole scaffold as a ligand that bound selectively to IIa IRES target and was confirmed as an inhibitor of in vitro viral translation. The screening approach outlined here provides an efficient method to discover HCV translation inhibitors that may provide leads for the development of novel antiviral therapies directed at the highly conserved IRES RNA.

The streptolidine lactam is an amino acid constituent of streptothricin antibiotics, which inhibit protein synthesis by targeting the bacterial ribosome but suffer from toxicity as a result of the basicity of the aminoimidazole scaffold in the natural products. On the basis of the streptolidine structure, we designed and synthesized oxazole analogs with six- and seven-membered lactam rings as building blocks for RNA-targeted ligands. These analogs benefit from a dense network of hydrogen-bond donors and acceptors within a rigid nonplanar heterocyclic system that has attenuated basicity.

The preparation and crystal structure of 8-chloro-1H-pyrrolo[2,3-b]phenazine5-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), β = 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. The synthesis, optical properties and crystal structure of the title compound is described.

International audience; Due to the in vitro antiplasmodial activity of leaf extracts from Vernonia fimbrillifera Less. (Asteraceae), a bioactivity-guided fractionation was carried out. Three sesquiterpene lactones were isolated, namely 8-(4’-hydroxymethacrylate) dehydromelitensin (1), onopordopicrin (2) and 8α-[4’-hydroxymethacryloyloxy]-4-epi-sonchucarpolide (3). Their structures were elucidated by spectroscopic methods (1D and 2D NMR and MS analyses) and by comparison with published data. The isolated compounds exhibited antiplasmodial activity with IC50 values ≤ 5 μg/mL. Cytotoxicity of the compounds against a human cancer cell line (HeLa) and a mouse lung epithelial cell line (MLE12) was assessed to determine selectivity. Compound 3 displayed promising selective antiplasmodial activity (SI > 10).

The compound 5, containing the novel heterocycle 2-oxa-4,7-diazabicyclo[3.3.1]non-3-ene, has been obtained in a synthetic approach toward oxazoles and 1,3-diazepanes of natural product-like complexity from cyclization and rearrangement of δ-lactam cyanamides. When this procedure was applied to a silyl-protected N-((3S,4S,5S)-4,5-dihydroxy-2-oxopiperidin-3-yl)cyanamide (2b) formation of the novel heterobicyclic scaffold 5 was observed along with the expected oxazole (3b) and diazepane (4b) products. The crystal structures of 5 and diazepane 4b are described. Compound 5 crystallized from methanol in the monoclinic system, P21 space group with unit cell parameters a = 15.3402(9), b = 7.2717(4), c = 22.5803(13), β = 106.8620(10) and a cell volume of 2410.5(2) A3. The synthesis and crystal structure of the title compound is described.

The title compound 1 has been prepared from the condensation product of a silyl protected amino-oxazoline lactam (3) and Cbz-protected l-tryptophan (4) after hydrogenolysis of the carboxybenzyl protection group. The synthesis and crystal structure of 1 is described. The compound crystallized from DMSO in the monoclinic system, P21 space group with unit cell parameters a = 9.4029(25), b = 6.2828(12), c = 20.681(45), and β = 96.505(16), Z = 2 and a cell volume of V = 1213.9(2) A3. In the crystal lattice, 1 forms an extensive hydrogen bonded framework consisting of interconnected dimeric chains. The synthesis and crystal structure of the title compound is described.

A sequence around the start codon of the mRNA of human thymidylate synthase (TS) folds into a secondary-structure motif in which the initiation site is sequestered in a metastable hairpin. Binding of the protein to its own mRNA at the hairpin prevents the production of TS through a translation-repression feedback mechanism. Stabilization of the mRNA hairpin by other ligands has been proposed as a strategy to reduce TS levels in anticancer therapy. Rapidly proliferating cells require high TS activity to maintain the production of thymidine as a building block for DNA synthesis. The crystal structure of a model oligonucleotide (TS1) that represents the TS-binding site of the mRNA has been determined. While fluorescence studies showed that the TS1 RNA preferentially adopts a hairpin structure in solution, even at high RNA concentrations, an asymmetric dimer of two hybridized TS1 strands was obtained in the crystal. The TS1 dimer contains an unusual S-­turn motif that also occurs in the ‘off’ state of the human ribosomal decoding site RNA.

Translation inhibitors of the 3,5-diamino-piperidine series act as aminoglycoside mimetics that inhibit bacterial growth. Here we show antibacterial SAR in the presence and absence of serum with a particular focus towards Pseudomonas aeruginosa.

The hepatitis C (HCV) internal ribosome entry site (IRES) element plays a central role in cap-independent translation of the viral genomic RNA. The unique conformation of IRES domain II is critical for 80S ribosomal assembly and initiation of viral translation. Here, the crystal structure of subdomain IIa of the HCV IRES has been determined at 2.3 {angstrom} resolution, revealing the positions of divalent metal ions and complex inter-strand interactions that stabilize the L-shaped conformation of the RNA. The presence of divalent metal ions was necessary for crystal formation. Magnesium ions occupy specific sites that appear to be critical for the formation of the folded conformation. Subdomain IIa also was crystallized in the presence of strontium, which improved the diffraction quality of the crystals and the ability to identify interactions of the RNA with metal ions and tightly bound water molecules. The hinge region and noncanonical G-U base-pair motifs are stabilized by divalent metal ions and provide unique structural features that are potential interaction sites for small-molecule ligands. The information obtained from the crystal structure provides a basis for structure-guided design of HCV translation inhibitors targeting disruption of ribosomal assembly.

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. The synthesis, structure–activity relationships (SAR), metabolic stability, and structure-based design approach for this new class of compounds are discussed.

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. The structure-activity relationship (SAR) associated with variation of the pyridazinone 2- and 6-substituents is discussed. The synthesis and metabolic stability of this new class of compounds are also described.

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.

2-Deoxystreptamine (2-DOS) aminoglycosides exert their antibiotic actions by binding to the A site of the 16S rRNA and interfering with bacterial protein synthesis. However, the molecular forces that govern the antitranslational activities of aminoglycosides are poorly understood. Here, we describe studies aimed at elucidating these molecular forces. In this connection, we compare the bactericidal, antitranslational, and rRNA binding properties of the 4,5-disubstituted 2-DOS aminoglycoside neomycin (Neo) and a conformationally restricted analog of Neo (CR-Neo) in which the 2′-nitrogen atom is covalently conjugated to the 5′′-carbon atom. The bactericidal potency of Neo exceeds that of CR-Neo, with this enhanced antibacterial activity reflecting a correspondingly enhanced antitranslational potency. Time-resolved fluorescence anisotropy studies suggest that the enhanced antitranslational potency of Neo relative to that of CR-Neo is due to a greater extent of drug-induced reduction in the mobilities of the nucleotides at positions 1492 and 1493 of the rRNA A site. Buffer- and salt-dependent binding studies, coupled with high-resolution structural information, point to electrostatic contacts between the 2′-amino functionality of Neo and the host rRNA as being an important modulator of 1492 and 1493 base mobilities and therefore antitranslational activities.

Conformational transitions in ribosomal decoding-site RNA have previously been monitored by replacement of flexible adenine bases with fluorescent 2-aminopurine (2AP). Here, we have studied pteridine base analogues 3-methylisoxanthopterin (3MI) and 6-methylisoxanthopterin (6MI) as fluorescent labels for the investigation of conformational changes in the decoding site that are triggered by ligand binding. The higher quantum yield of 3MI and 6MI along with their relatively red-shifted excitation and emission wavelengths renders the pteridines interesting alternatives to 2AP for the establishment of ligand-binding assays for RNA based on fluorescence labelling.

The RNA genome of the hepatitis C virus (HCV) contains an internal ribosome entry site (IRES), which binds to the hostcell 40S ribosomal subunit and initiates protein translation in the absence of most initiation factors. Recruitment of the ribosomal subunit to the HCV RNA is driven by the high affinity of the IRES–40S interaction. The IRES sequence adopts a highly ordered secondary structure (Figure 1a). The three-dimensional architecture of the IRES is dominated by independently folding RNA domains. Structures of IRES–40S complexes have been studied by cryo-electron microscopy (cryo-EM) revealing the overall shape of the RNA. Higher-resolution structures of individual subdomains, including II and IIIa–e, have been determined by crystallography and NMR spectroscopy. Cryo-EM studies revealed that the domain II, which plays an important structural role in HCV translation, adopts an L-shaped conformation that directs the apical hairpin loop IIb to overlap with the ribosomal E site in the proximity of the P site. Binding of domain II induces a conformational change in the 40S head and closes the messenger RNA (mRNA) binding cleft. NMR studies suggested that subdomain IIa might be a flexible hinge whose bent state is stabilized by binding of divalent metal ions. To investigate the molecular architecture of the IRES domain II kink and its metal-ion-dependent stabilization, we have used X-ray crystallography and structure-guided incorporation of fluorescent labels. For X-ray crystal structure determination (see Figure S4 and Table S1 in the Supporting Information), an oligonucleotide (IIa-1) was used that contained residues 49–69 and 100–115 of the HCV IRES (Figure 1). The overall structure of the RNA revealed a bent architecture for the IIa subdomain. The two stems that are flanking the internal bulge are arranged at a right angle (Figure 1b). The upper stem (residues 58–69/100–110) forms a continuous helix that contains both standard and noncanonical base pairs that adopt cis-Watson–Crick geometries. U106 is looped out from the stem to allow continuous stacking of the flanking base pairs. The arrangement of residues of the internal bulge introduces a right-angled bend in the RNA

Syntheses of dehydroalanine derivatives via a solid-support route, starting from selenocystein, and via conventional solution phase chemistry are described along with initial biological testing. The target compounds were designed as mimetics of the dehydroalanine side chain of the macrocyclic antibiotic thiostrepton that acts on the bacterial ribosome.

RNA recognition by natural aminoglycoside antibiotics depends on the 2-deoxystreptamine (2-DOS) scaffold which participates in specific hydrogen bonds with the ribosomal decoding-site target. Three-dimensional structure information has been used for the design of azepane-monoglycosides, building blocks for novel antibiotics in which 2-DOS is replaced by a heterocyclic scaffold. Azepane-glycosides showed target binding and translation inhibition in the low micromolar range and inhibited growth of Staphylococcus aureus, including aminoglycoside-resistant strains.

In the 1950s Corynebacterium glutamicum was found to be a very efficient producer of L-glutamic acid. Since this time biotechnological processes with bacteria of the species Corynebacterium developed to be among the most important in terms of tonnage and economical value. L-Glutamic acid and L-lysine are bulk products nowadays. L-Valine, L-isoleucine, L-threonine, L-aspartic acid and L-alanine are among other amino acids produced by Corynebacteria. Applications range from feed to food and pharmaceutical products. The growing market for amino acids produced with Corynebacteria led to significant improvements in bioprocess and downstream technology as well as in molecular biology. During the last decade big efforts were made to increase the productivity and to decrease the production costs. This review gives an overview of the world market for amino acids produced by Corynebacteria. Significant improvements in bioprocess technology, i.e. repeated fed batch or continuous production are summarised. Bioprocess technology itself was improved furthermore by application of more sophisticated feeding and automatisation strategies. Even though several amino acids developed towards commodities in the last decade, side aspects of the production process like sterility or detection of contaminants still have increasing relevance. Finally one focus of this review is on recent developments in downstream technology.

An efficient and highly versatile synthesis of two libraries 1(x,y) and 2-Ar(x,y,z) or 2-R 2 (x,y,w) based on the privileged benzimidazole scaffold is described. Our design is aimed at obtaining molecules, biased for binding to RNA targets, by incorporating functionalities, which are frequently found in natural RNA-ligands. The library construction was realized with the use of SPOS in high average yields and purity. Monitoring and quantitation of intermediates and final products were performed by the use of NMR spectroscopy using DMFu as an internal standard.

The ribosomal decoding site is the target of aminoglycoside antibiotics that specifically recognize an internal loop RNA structure. We synthesized RNA-targeted 2,5-dideoxystreptamine-4-amides in which a sugar moiety in natural aminoglycosides is replaced by heterocycles.

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., We used crystallography and site-specific cross-linking to study interacting regions of the human thymidylate synthase and its cognate mRNA, which form a complex that represses translation of the enzyme and is implicated in resistance of tumours to antimetabolite chemotherapy.

By the use of different Corynebacterium glutamicum strains move than 1.4 million tons of amino acids, mainly L-glutamate and L-lysine, are produced per year. A project was started recently to elucidate the complete DNA sequence of this bacterium. In this communication we describe an approach to analyze the C, glutamicum proteome, based on this genetic information, by a combination of two-dimensional (2-D) gel electrophoresis and protein identification via microsequencing or mass spectrometry. We used these techniques to resolve proteins of C, glutamicum with the aim to establish 2-D protein maps as a tool for basic microbiology and for strain improvement. In order to analyze the C. glutamicum proteome, methods were established to fractionate the C. glutamicum proteins according to functional entities, i.e., cytoplasm, membranes, and cell wall. Protein spots of the cytoplasmic and membrane fraction were identified by N-terminal sequencing, immunodetection, matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-mass spectrometry (ESI-MS). Additionally, a protocol to analyze proteins secreted by C. glutamicum was established. Approximately 40 protein spots were observed on silver-stained 2-D gels, 12 of which were identified.

We have used NMR spectroscopy to determine the solution structure of a complex between an oligonucleotide derived from stem IIB of the Rev responsive element (RRE-IIB) of HIV-1 mRNA and an in vivo selected, high affinity binding Arg-rich peptide. The peptide binds in a partially alpha-helical conformation into a pocket within the RNA deep groove. Comparison with the structure of a complex between an alpha-helical Rev peptide and RRE-IIB reveals that the sequence of the bound peptide determines the local conformation of the RRE peptide binding site. A conformational switch of an unpaired uridine base was revealed; this points out into the solvent in the Rev peptide complex, but it is stabilized inside the RNA deep groove by stacking with an Arg side chain in the selected peptide complex. The conformational switch has been visualized by NMR chemical shift mapping of the uridine H5/H6 atoms during a competition experiment in which Rev peptide was displaced from RRE-IIB by the higher affinity binding selected peptide.

In many steps of gene replication and expression, RNA molecules participate as key players, which renders them attractive targets for therapeutic intervention. While the function of nucleic acids as carriers of genetic material is based on their sequence, a number of important RNAs are involved in processes that depend on the defined three-dimensional structures of these molecules. As for proteins, numerous complex folds of RNA exist. The development of drugs that bind specifically to RNA folds opens exciting new ways to expand greatly the existing repertoire of protein-targeted therapeutics. Most functions of RNAs involve interactions with proteins that contain RNA-binding domains. Effector molecules targeted at RNA may either alter the functional three-dimensional structure of the nucleic acid, so the interaction with proteins is thereby inhibited or enhanced, or, as interface inhibitors, they may directly prevent the formation of competent RNA-protein complexes. While the same tools used for the design of protein-targeted drugs may be considered for studying effectors binding to nucleic acids, the differences between proteins and RNAs in the forces which dominate their three-dimensional folding call for novel drug design strategies. In the present review, I will outline how our rapidly expanding knowledge of RNA three-dimensional structure and function facilitates rational approaches to develop RNA-binding compounds. Putative RNA targets for therapeutic intervention will be discussed along with recent advances in understanding RNA-small molecule and RNA-protein interactions.

RNA molecules are the only known molecules which possess the double property of being depository of genetic information, like DNA, and of displaying catalytic activities, like protein enzymes. RNA molecules intervene in all steps of gene expression and in many other biological activities. Like proteins, RNAs achieve those biological functions by adopting intricate three-dimensional folds and architectures. Further, as in protein sequences, RNA sequences contain signatures specific for three - dimensional motifs which participate in recognition and binding. In regulatory pathways, RNA molecules exist in equilibria between transient structures differentially stabilized by effectors such as proteins or cofactors. Therefore, RNA molecules display their potential as drug targets on different levels, namely in three-dimensional folds, in structural equilibria and in RNA-protein interfaces. Several examples will be described together with the already available techniques for combinatorial synthesis and high-throughput screening of potential drug and target RNA molecules.

Proteins strongly synthesized in Corynebacterium glutamicum during nitrogen restriction were examined by two-dimensional gel electrophoresis and microsequencing. Two main groups of enzymes were identified beside miscellaneous proteins, enzymes involved (i) in protein synthesis, and (ii) in carbon metabolism. Biochemical measurements revealed an increase of oxygen consumption during nitrogen starvation, indicating an enhanced energy demand of the cells. By Northern hybridizations, an increased transcription for the gap and fda genes upon nitrogen deprivation was shown.

Nucleic acid molecules play crucial roles in diverse biological processes including the storage, transport, processing, and expression of the genetic information. Nucleic acid aptamers are selected in vitro from libraries containing random sequences of up to a few hundred nucleotides. Selection is based on the ability to bind ligand molecules with high affinity and specificity. Three-dimensional structures have been determined at high resolution for a number of aptamers in complex with their cognate ligands. Structures of aptamer complexes reveal the key molecular interactions conferring specificity to the aptamer-ligand association, including the precise stacking of flat moieties, specific hydrogen bonding, and molecular shape complementarity. These basic principles of discriminatory molecular interactions in aptamer complexes parallel recognition events central to many cellular processes involving nucleic acids.

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.

Retroviruses encapsidate their genome as a dimer of homologous RNA molecules noncovalently linked close to their 5' ends. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) RNA is a hairpin structure that contains in the loop a 6-nt self-complementary sequence flanked by two 5' and one 3' purines. The self-complementary sequence, as well as the flanking purines, are crucial for dimerization of HIV-1 RNA, which is mediated by formation of a "kissing-loop" complex between the DIS of each monomer. Here, we used chemical modification interference, lead-induced cleavage, and three-dimensional modeling to compare dimerization of subtype A and B HIV-1 RNAs. The DIS loop sequences of these RNAs are AGGUGCACA and AAGCGCGCA, respectively. In both RNAs, ethylation of most but not all phosphate groups in the loop and methylation of the N7 position of the G residues in the self-complementary sequence inhibited dimerization. These results demonstrate that small perturbations of the loop structure are detrimental to dimerization. Conversely, methylation of the N1 position of the first and last As in the loop were neutral or enhanced dimerization, a result consistent with these residues forming a noncanonical sheared base pair. Phosphorothioate interference, lead-induced cleavage, and Brownian-dynamics simulation revealed an unexpected difference in the dimerization mechanism of these RNAs. Unlike subtype B, subtype A requires binding of a divalent cation in the loop to promote RNA dimerization. This difference should be taken into consideration in the design of antidimerization molecules aimed at inhibiting HIV-1 replication.

Molecular dynamics simulations of the RNA-binding domain of the U1A spliceosomal protein in complex with its cognate RNA hairpin, performed at low and high ionic strength in aqueous solution, suggest a pathway for complex dissociation. First, cations condense around the RNA and compete with the protein for binding sites. Then solvated ions specifically destabilize residues at the RNA-protein interface. For a discrete cluster of residues at the complex interface, the simulations reveal an increased deviation from the crystal structure at high salt concentrations while the remaining protein scaffold is stabilized under these conditions. The microscopic picture of salt influence on the complex suggests guidelines for rational design of interface inhibitors targeted at RNA-protein complexes.

A screen for compounds that block a bacterial biosynthetic pathway has uncovered an antibiotic lead that shuts off pathogen growth by targeting a molecular switch in a regulatory RNA structure. See Article p.672 The urgent need for new antibiotics is well recognized. Terry Roemer and colleagues at Merck now describe a new synthetic antibiotic, directed against a bacterial riboswitch. Riboswitches are stretches of non-coding RNA whose structure is affected by a ligand — usually one related to the function of the protein encoded by the riboswitch-containing gene. The new drug, ribocil, blocks the flavin mononucleotide riboswitch-mediated expression of the ribB gene required for riboflavin biosynthesis. Ribocil inhibits bacterial cell growth and is effective in treating a bacterial infection in a mouse model.

Background: Metal ions participate in the three-dimensional folding of RNA and provide active centers in catalytic RNA molecules. The positions of metal ions are known for a few RNA structures determined by X-ray crystallography. In addition to the crystallographically identified sites, solution studies point to many more metal ion binding sites around structured RNAs. Metal ions are also present in RNA structures determined by nuclear magnetic resonance (NMR) spectroscopy, but the positions of the ions are usually not revealed. Results: A novel method for predicting metal ion binding sites in RNA folds has been successfully applied to a number of different RNA structures. The method is based on Brownian-dynamics simulations of cations diffusing under the influence of random Brownian motion within the electrostatic field generated by the static three-dimensional fold of an RNA molecule. In test runs, the crystallographic positions of Mg2+ ions were reproduced with deviations between 0.3 and 2.7 A for several RNA molecules for which X-ray structures are available. In addition to the crystallographically identified metal ions, more binding sites for cations were revealed: for example, tRNAs were shown to bind more than ten Mg2+ ions in solution. Predictions for metal ion binding sites in four NMR structures of RNA molecules are discussed. Conclusions: The successful reproduction of experimentally observed metal ion binding sites demonstrates the efficiency of the prediction method. A promising application of the method is the prediction of cation-binding sites in RNA solution structures, determined by NMR.

A variety of drugs inhibit biological key processes by binding to a specific RNA component. We focus here on the well-analysed hammerhead ribozyme RNA that is inhibited by aminoglycoside antibiotics, a process considered as a paradigm for studying drug/RNA interactions. With insight gained from molecular dynamics simulations of the ribozyme in the presence of Mg 2a identified by crystallography and of aminoglycosides in solution, a general model for aminoglycoside binding to RNA is proposed. A striking structurally based complementarity between the charged ammonium groups of the aminoglycosides and the metal binding sites in the hammerhead was uncovered. Despite dynamical flexibility of the aminoglycosides, several of the intramolecular distances between the charged ammonium groups of the drugs were found to be rather constant. Intramolecular ammonium distances of the aminoglycosides span ranges similar to the interionic distances between Mg 2a in the hammerhead. Successful docking of aminoglycosides to the hammerhead ribozyme could be achieved by positioning the ammonium groups at the sites occupied by Mg 2a . The covalently linked ammonium groups of the aminoglycosides are thus able to complement in space the negative electrostatic potential created by a three-dimensional RNA fold. Consequently, it is suggested that aminoglycoside-derived sugars could constitute a basic set of yardstick synthons ideal for rational and combinatorial synthesis of drugs targeted at biologically relevant RNA folds. # 1998 Academic Press Limited

Among saccharides, the antibiotics of the aminoglycoside family are the best-studied class of molecules interacting with RNA. By binding to RNA targets, aminoglycosides act as inhibitors of protein biosynthesis, they interfere with protein-RNA interaction of retroviral regulatory elements, and they inhibit the catalytic action of ribozymes. Here, we survey the available data on molecular structural details of aminoglycoside-RNA interaction.

Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein. Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology. Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS. Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question. Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro. We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner. Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.

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) A, b = 14.0128(4) A, c = 8.8147(3) A, β = 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. The crystal structure of the title compound is described free and in complex with its viral target.