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Cost-effective, fast, and reliable DNA sequencing can be enabled by advances in nanopore-based methods, such as the use of atomically thin graphene membranes. However, strong interaction of DNA bases with graphene leads to undesirable effects such as sticking of DNA strands to the membrane surface. While surface functionalization is one way to counter this problem, here, we present another solution based on a heterostructure nanopore system, consisting of a monolayer of graphene and hexagonal boron nitride (hBN) each. Molecular dynamics studies of DNA translocation through this heterostructure nanopore revealed a surprising and crucial influence of the heterostructure layer order in controlling the base specific signal variability. Specifically, the heterostructure with graphene on top of hBN had nearly 3-10× lower signal variability than the one with hBN on top of graphene. Simulations point to the role of differential underside sticking of DNA bases as a possible reason for the observed influence of the layer order. Our studies can guide the development of experimental systems to study and exploit DNA translocation through two-dimensional heterostructure nanopores for single molecule sequencing and sensing applications.

We present the rapid biophysical characterization of six previously reported putative G-quadruplex-forming RNAs from the 5'-untranslated region (5'-UTR) of silvestrol-sensitive transcripts for investigation of their secondary structures. By NMR and CD spectroscopic analysis, we found that only a single sequence-[AGG] 2 [CGG] 2 C-folds into a single well-defined G-quadruplex structure. Sequences with longer poly-G strands form unspecific aggregates, whereas CGG-repeat-containing sequences exhibit a temperature-dependent equilibrium between a hairpin and a G-quadruplex structure. The applied experimental strategy is fast and provides robust readout for G-quadruplex-forming capacities of RNA oligomers., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The ubiquitous influence of double stranded RNAs in biological events makes them imperative to gather data based on specific binding procedure of small molecules to various RNA conformations. Particular interest may be attributed to situations wherein small molecules target RNAs altering their structures and causing functional modifications. The main focus of this study is to delve into the interactive pattern of two small molecule phenothiazinium dyes, methylene blue and new methylene blue, with three duplex RNA polynucleotides-poly(A).poly(U), poly(C).poly(G) and poly(I).poly(C) by spectroscopic and molecular modeling techniques. Analysis of data as per Scatchard and Benesi-Hildebrand methodologies revealed highest affinity of these dyes to poly(A).poly(U) and least to poly(I).poly(C). In addition to fluorescence quenching, viscometric studies also substantiated that the dyes follow different modes of binding to different RNA polynucleotides. Distortion in the RNA structures with induced optical activity in the otherwise optically inactive dye molecules was evidenced from circular dichroism results. Dye-induced RNA structural modification occurred from extended conformation to compact particles visualized by atomic force microscopy. Molecular docking results revealed different binding patterns of the dye molecules within the RNA duplexes. The novelty of the present work lies towards a new contribution of the phenothiazinium dyes in dysfunctioning double stranded RNAs, advancing our knowledge to their potential use as RNA targeted small molecules., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Alginate, the most abundant carbohydrate in brown macroalgae, is widely used in the food and pharmaceutical industries. Recently, alginate has attracted increasing attention, as it may serve as an alternative biomass for the production of biofuel. The degradation of alginate into monomeric units is the prerequisite for bioethanol production. All known oligoalginate lyases belong to the polysaccharide lyase (PL) family 7, 14, 15 and 17, and most of them preferred to degrade the polyM blocks to yield 4-deoxy-l-erythro-5-hexoseulose uronic acid as the primary product. In this study, we cloned an oligoalginate lyase gene, oalS6, from Shewanella sp. Kz7 and expressed it in Escherichia coli. The PL family 6 oligoalginate lyase (OalS6) has no significant sequence similarity with other known oligoalginate lyases. OalS6 contains a chondroitinase-like domain and was assigned to the PL family 6. This lyase is an exo-type oligoalginate lyase and prefer to depolymerize polyG block into 2, 4, 5, 6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid. All of these results indicate that OalS6 is a novel oligoalginate lyase that is structurally and functionally different from other known oligoalginate lyases. This finding provides new insights into the development of biofuel processing biotechnologies from seaweed., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Delivery to intracellular target sites is still one of the main obstacles in the development of peptide nucleic acids (PNAs) as antisense-antigene therapeutics. Here, we designed a self-assembled oligonucleotide scaffold that included a central complementary region for self-assembly and lateral regions complementing the PNAs. Assembly of cell-penetrating peptide (CPP)-PNAs on the scaffold significantly promoted endocytosis of PNAs by at least 10-fold in cell cultures, particularly for scaffolds in which the central complementary region was assembled by poly(guanine) and poly(cytosine). The antisense activity of CPP-PNAs increased by assembly on the scaffold and was further enhanced after co-assembly with endosomolytic peptide (EP)-PNA. This synergistic effect was also observed following the assembly of antigene CPP-PNAs\EP-PNAs on the scaffold. However, antigene activity was only observed by targeting episomal viral DNA or transfected plasmids, but not the chromosome in the cell cultures. In conclusion, assembly on oligonucleotide scaffolds significantly enhanced the antisense-antigene activity of PNAs by promoting endocytosis and endosomal escape. This oligonucleotide scaffold provided a simple strategy for assembly of multiple functional peptide-PNA conjugates, expanding the applications of PNAs and demonstrating the potential of PNAs as antiviral therapeutics.

Lysosomes can degrade various biological macromolecules, including nucleic acids, proteins and lipids. Recently, we identified novel nucleic acid-degradation systems termed RNautophagy/DNautophagy (abbreviated as RDA), in which RNA and DNA are directly taken up by lysosomes in an ATP-dependent manner and degraded. We also found that a lysosomal membrane protein, LAMP2C, the cytoplasmic region of which binds to RNA and DNA, functions, at least in part, as an RNA/DNA receptor in the process of RDA. However, it has been unclear whether RDA possesses selectivity for RNA/DNA substrates and the RNA/DNA sequences that are recognized by LAMP2C have not been determined. In the present study, we found that the cytosolic region of LAMP2C binds to poly-G/dG, but not to poly-A/dA, poly-C/dC, poly-dT or poly-U. Consistent with this binding activity, poly-G/dG was transported into isolated lysosomes via RDA, while poly-A/dA, poly-C/dC, poly-dT and poly-U were not. GGGGGG or d(GGGG) sequences are essential for the interaction between poly-G/dG and LAMP2C. In addition to poly-G/dG, G/dG-rich sequences, such as a repeated GGGGCC sequence, interacted with the cytosolic region of LAMP2C. Our findings indicate that RDA does possess selectivity for RNA/DNA substrates and that at least some consecutive G/dG sequence(s) can mediate RDA., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Binding of a novel cationic porphyrin-imidazophenazine conjugate, TMPyP(3+)-ImPzn, to four-stranded poly(G) was investigated in aqueous solutions of neutral pH under near physiological ionic conditions using absorption, polarized fluorescent spectroscopy and fluorescence titration techniques. In absence of the polymer the conjugate folds into stable internal heterodimer with stacking between the porphyrin and phenazine chromophores. Binding of TMPyP(3+)-ImPzn to poly(G) is realized by two competing ways. At low polymer-to-dye ratio (P/D < 6) outside electrostatic binding of the cationic porphyrin moieties of the conjugate to anionic polynucleotide backbone with their self-stacking is predominant. It is accompanied by heterodimer dissociation and distancing of phenazine moieties from the polymer. This binding mode is characterized by strong quenching of the conjugate fluorescence. Increase of P/D results in the disintegration of the porphyrin stacks and redistribution of the bound conjugate molecules along the polymer chain. At P/D > 10 another binding mode becomes dominant, embedding of TMPyP(3+)-ImPzn heterodimers into poly(G) groove as a whole is occurred.

Studies on the molecular aspects of alkaloid-RNA complexation are of prime importance for the development of rational RNA targeted drug design strategies. Towards this goal, the binding aspects of three novel 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs to four single stranded ribonucleotides, poly(G), poly(I), poly(C) and poly(U), were studied for the first time employing multifaceted biophysical tools. Absorbance and fluorescence studies revealed that these analogs bound non-cooperatively to poly(G) and poly(I) with binding affinities remarkably higher than berberine. The binding of these analogs to poly(U) and poly(C) was weaker in comparison to poly(G) and poly(I) but were one order higher in comparison to berberine. Quantum efficiency values revealed that energy transfer occurred from the RNA bases to the analogs upon complexation. The binding was dominated by large positive entropic contributions and small but favorable enthalpic contributions. Salt dependent studies established that the binding was dominated by hydrophobic forces that contributed around 90% of the total standard molar Gibbs energy. The chain length of the substitution at the 9-position was found to be critical in modulating the binding affinities. These results provide new insights into the binding efficacy of these novel berberine analogs to single stranded RNA sequences., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Deadenylation is the first and rate-limiting step during turnover of mRNAs in eukaryotes. In the yeast, Saccharomyces cerevisiae, two distinct 3'-5' exonucleases, Pop2p and Ccr4p, have been identified within the Ccr4-NOT deadenylase complex, belonging to the DEDD and Exonuclease-Endonuclease-Phosphatase (EEP) families, respectively. Ngl3p has been identified as a new member of the EEP family of exonucleases based on sequence homology, but its activity and biological roles are presently unknown. Here, we show using in vitro deadenylation assays on defined RNA species mimicking poly-A containing mRNAs that yeast Ngl3p is a functional 3'-5' exonuclease most active at slightly acidic conditions. We further show that the enzyme depends on divalent metal ions for activity and possesses specificity towards poly-A RNA similar to what has been observed for cellular deadenylases. The results suggest that Ngl3p is naturally involved in processing of poly-adenylated RNA and provide insights into the mechanistic variations observed among the redundant set of EEP enzymes found in yeast and higher eukaryotes.

A 20-kDa ribonuclease (RNase) was purified from fresh fruiting bodies of cultured Schizophyllum commune mushrooms. The RNase was not adsorbed on Affi-gel blue gel but adsorbed on DEAE-cellulose and CM-cellulose. It exhibited maximal RNase activity at pH 6.0 and 70°C. It demonstrated the highest ribonucleolytic activity toward poly (U) (379.5 μ/mg), the second highest activity toward poly (C) (244.7 μ/mg), less activity toward poly (A) (167.4 μ/mg), and much weaker activity toward poly (G) (114.5 μ/mg). The RNase inhibited HIV-1 reverse transcriptase with an IC(50) of 65 μM. No effect on [(3)H-methyl]-thymidine uptake by lymphoma MBL2 cells and leukemia L1210 cells was observed at 100 μM concentration of the RNase. A comparison of RNases from S. commune and Volvariella volvacea revealed that they demonstrated some similarities in N-terminal amino acid sequence, optimum pH and polyhomoribonucleotide specificity. However, some differences in chromatographic behavior and molecular mass were observed.

Elucidation of the molecular aspects of small molecule-RNA complexation is of prime importance for rational RNA targeted drug design strategies. Towards this, the interaction of the cytotoxic plant alkaloid sanguinarine to three double stranded ribonucleic acids, poly (A).poly(U), poly(I).poly(C) and poly(C).poly(G) was studied using various biophysical and thermodynamic techniques. Absorbance and fluorescence studies showed that the alkaloid bound cooperatively to these RNAs with binding affinities of the order 10(4) M(-1). Fluorescence quenching and hydrodynamic studies gave evidence for intercalation of sanguinarine to these RNA duplexes. Isothermal titration calorimetric studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the affinity constants derived were in agreement with the overall binding affinity values obtained from spectroscopic data. The binding of sanguinarine stabilized the melting of poly(A). poly(U) and poly(I).poly(C) and the binding data evaluated from the melting data were in agreement with that obtained from other techniques. The overall binding affinity of sanguinarine to these double stranded RNAs varied in the order, poly(A).poly(U) > poly(I).poly(C) >> poly(C).poly(G). The temperature dependence of the enthalpy changes afforded negative values of heat capacity changes for the binding of sanguinarine to poly(A).poly(U) and poly(I).poly(C), suggesting substantial hydrophobic contribution in the binding process. Further, enthalpy-entropy compensation phenomena was also seen in poly(A).poly(U) and poly(I).poly(C) systems that correlated to the strong binding involving a multiplicity of weak noncovalent interactions compared to the weak binding with poly(C).poly(G). These results further advance our understanding on the binding of small molecules that are specific binders to double stranded RNA sequences.

The ability of guanine-rich sequences to form quadruplex structures in telomeres for example is important in a number of biological processes such as aging, carcinogenesis and gene regulation. Ionizing radiation can cause damage to guanine moieties that can affect the stability or formation of the guanine quadruplex structures. In addition, the mechanisms of formation of these radiation damages in quadruplex structures may be different from those that occur in single- or double-stranded conformations. We have studied the quantitative aspects of the radiation induced formation of 8-hydroxy-2'-guanine base modifications and unaltered guanine base release in single-, double- and four-stranded conformations of polyriboguanylic acid as a model of guanine-rich sequences in telomere-like structures. The results show that the strandedness of guanine-rich sequences is an important variable in the observed yields of these base damages and suggests that telomere-like structures with G-quadruplexes will be relatively more radiosensitive than the other regions of duplex DNA. Hydroxyl radicals are the major reactive species that produce the DNA damage, although the presence of oxygen significantly reduces their radiation yields for all conformations of polyriboguanylic acid and changes the proportions of the yields of the various damages among the polymer conformations.

Design and synthesis of new small molecules binding to double-stranded RNA necessitate complete understanding of the molecular aspects of the binding of many existing molecules. Toward this goal, in this work we evaluated the biophysical aspects of the interaction of a DNA intercalator (proflavine) and a minor groove binder (hoechst 33258) with two polymorphic forms of polyCG, namely, the right-handed Watson-Crick base paired A-form and the left-handed Hoogsteen base paired H(L)-form, by absorption, fluorescence, and viscometry experiments. The energetics of the interaction of these molecules with the RNA structures has also been elucidated by isothermal titration calorimetry (ITC). Results suggest that proflavine strongly intercalates in both forms of polyCG, whereas hoechst shows mainly groove-binding modes. The binding of both drugs to both forms of RNA resulted in significant conformational change to the RNA structure with the bound molecules being placed in the chiral RNA helix. ITC profiles for both proflavine and hoechst show two binding sites. Binding of proflavine to both forms of RNA is endothermic and entropy driven in the first site and exothermic and enthalpy driven in the second site, whereas hoechst binding to both forms of RNA is exothermic and enthalpy driven in the first site and endothermic and entropy driven in the second site. This study suggests that the binding affinity characteristics and energetics of interaction of these DNA binding molecules with the RNA conformations are significantly different and may serve as data for future development of effective structure-selective RNA-based drugs.

It was found recently that Hoechst 33258, a dsDNA fluorescent dye used in cytological studies, is an efficient inhibitor of the interaction of TATA-box-binding protein with DNA, DNA topoisomerase I, and DNA helicases. In addition it proved to be a radioprotector. Biological activity of Hoechst 33258 may be associated with dsDNA complexes of not only monomeric, but also dimeric type. In this work, the Hoechst 33258 interaction with poly(dG-dC).poly(dG-dC) was studied using UV-vis and fluorescent spectroscopy, circular and flow-type linear dichroism. It was found that Hoechst 33258 formed with poly(dG-dC).poly(dG-dC) complexes of three types, namely, monomeric, dimeric, and, apparently, tetrameric, and their spectral properties were studied. Complexes of monomeric and dimeric types competed with distamycin A, a minor groove ligand, for binding to poly(dG-dC).poly(dG-dC). We proposed that Hoechst 33258 both monomers and dimers form complexes of the external type with poly(dG-dC).poly(dG-dC) from the side of the minor groove.

The prevailing paradigm ascribes activation of Toll-like receptor 9 (TLR9) to the detection of CpG-motifs within pathogen derived DNA. However, new work ties natural phospho-diester (PD) DNA recognition by TLR9 to the detection of the DNA sugar backbone 2' deoxyribose. PD 2' deoxyribose homopolymers lacking DNA bases (abasic) are shown to act as TLR9 agonist while abasic phospho-thioate (PS) 2' deoxyribose functions as TLR9 antagonist. Alignment of bases to PD 2' deoxyribose enhanced its TLR9 agonistic function, while only CpG-motifs introduced to inhibitory PS 2' deoxyribose converted the antagonistic activity into powerful agonistic function. These new data thus restrict the CpG-motif dependency of TLR9 activation to the promising group of immunopharmacons that are based on PS modified synthetic DNA. They also show that natural PD DNA drives TLR9 activation sequence-independently as is the case for ds RNA recognizing TLR3 and ss RNA recognizing TLR7 and TLR8. Thus evolutionary pressure might have exiled nucleic acid recognizing TLRs such as TLR9 to endosomes in order to avoid activation by host (self) derived nucleic acids.

Aggregates of the self-assembling peptide EAK16II or EAK16IV and oligodeoxynucleotides (ODNs) were prepared, and their stability upon diluting the solution was investigated by UV-vis spectroscopy. The aggregates prepared at pH 4 and pH 7 did not dissociate after the solution was diluted 5- and 10-fold. The resistance against Escherichia coli exonuclease I of the ODN located in the EAK-ODN aggregates was studied by fluorescence resonance energy transfer (FRET) after the ODN had aggregated with EAK16II or EAK16IV at pH 4 or pH 7. The effect that the peptide sequence, peptide concentration, pH, and centrifugation had on protecting the aggregated ODN against nuclease degradation was investigated. Significant nuclease resistance was obtained after the EAK-ODN aggregates had been prepared at pH 4, with an EAK16IV concentration greater than a threshold value, and ensuring that the solution was not centrifuged immediately after sample preparation. Centrifuging the EAK16IV-ODN solution immediately after sample preparation resulted in the loss of this nuclease protection. However, if the solution of EAK-ODN aggregates was centrifuged 24 h after sample preparation, the nuclease protection afforded by the EAK16IV-ODN aggregates to the ODN was maintained even after being subject to a 10-fold dilution and up to 4 rounds of centrifugation over 4 days.

Double-stranded RNA homopolymer poly(rC).poly(rG) has been used as a new pH-sensitive drug carrier. The poly(rC).poly(rG) had proton buffering capacity around pH 6, owing to protonation of cytosine, as determined by acid-base titration. By circular dichroism measurement, the protonation caused conformational change of the RNA. The poly(rC).poly(rG) and doxorubicin (Dox), as an anticancer drug, formed the complexes which released the drugs at endosomal pH. The resulting complex exhibited higher anticancer activity than the Dox alone. These results result suggest that the poly(rC).poly(rG) is a promising biopolymer for a new class of pH-sensitive drug carriers.

A key step in the rational design of new RNA binding small molecules necessitates a complete elucidation of the molecular aspects of the binding of existing molecules to RNA structures. This work focuses towards the understanding of the interaction of a DNA intercalator, quinacrine and a minor groove binder 4',6-diamidino-2-phenylindole (DAPI) with the right handed Watson-Crick base paired A-form and the left-handed Hoogsteen base paired H(L)-form of poly(rC).poly(rG) evaluated by multifaceted spectroscopic and viscometric techniques. The energetics of their interaction has also been elucidated by isothermal titration calorimetry. Results of this study converge to suggest that (i) quinacrine intercalates to both A-form and H(L)-form of poly(rC).poly(rG); (ii) DAPI shows both intercalative and groove-binding modes to the A-form of the RNA but binds by intercalative mode to the H(L)-form. Isothermal calorimetric patterns of quinacrine binding to both the forms of RNA and of DAPI binding to the H(L)-form are indicative of single binding while the binding of DAPI to the A-form reveals two kinds of binding. The binding of both the drugs to both conformations of RNA is exothermic; while the binding of quinacrine to both conformations and DAPI to the A-form (first site) is entropy driven, the binding of DAPI to the second site of A-form and H(L)-conformation is enthalpy driven. Temperature dependence of the binding enthalpy revealed that the RNA-ligand interaction reactions are accompanied by small heat capacity changes that are nonetheless significant. We conclude that the binding affinity characteristics and energetics of interaction of these DNA binding molecules to the RNA conformations are significantly different and may serve as data for the development of effective structure selective RNA-based antiviral drugs.

Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus with a single-stranded RNA genome containing non-coding regions (NCRs) at its 5' and 3'-ends. The NCRs have flavivirus-conserved sequences that are important for virus replication. Here we describe DNAzymes (Dzs) that cleave the RNA sequence of the 3'-NCR of JEV genome in vitro. The nuclease-resistant Dzs, containing phosphorothioate linkages, were efficiently taken up by mouse neuronal and glial cells, and addition of a continuous stretch of 10 guanosine residues (poly-(G)(10)) to the 3'-end of a Dz led to its enhanced delivery to cells containing scavenger receptors (ScRs). These novel Dzs inhibited JEV replication in cultured mouse cells of neuronal and macrophage origin. JEV is a neurotropic virus that actively replicates in mouse brain. Here we show that intra-cerebral (i.c.) administration of a poly-(G)(10)-tethered, phosphorothioated Dz in JEV-infected mice led to more than 99.99% inhibition of virus replication in brain, resulting in a dose-dependent extended lifespan or complete recovery of the infected animals. This is the first report of in vivo application of a Dz to control a virus infection in an animal model.

The Al(III)-salophen complex 1 exhibited strong spectroscopic changes specifically upon addition of polyG and GpG, while double stranded DNA and RNA, and single stranded polyA, polyU and polyC induced negligible spectral changes of 1. Titrations with mono-nucleotides yielded no spectroscopic changes, revealing that there must be at least two consecutive guanines in single stranded oligonucleotide structure for a measurable spectroscopic change of 1. Preliminary results show that 1 has moderate antiproliferative effect on a number of human tumour cell lines.

G-Rich sequences are known to form four-stranded structures that are based on stacks of G-quartets, and sequences with the potential to adopt these structures are common in eukaryotic genomes. However, there are few rules for predicting the relative stability of folded complexes that are adopted by sequences with different-length G-tracts or variable-length linkers between them. We have used thermal melting, circular dichroism, and gel electrophoresis to examine the topology and stability of intramolecular G-quadruplexes that are formed by sequences of the type d(GnT)4 and d(GnT2)4 (n = 3-7) in the presence of varying concentrations of sodium and potassium. In the presence of potassium or sodium, d(GnT)4 sequences form intramolecular parallel complexes with the following order of stability: n = 3 > n = 7 > n = 6 > n = 5 > n = 4. d(G3T)4 is anomalously stable. In contrast, the stability of d(GnT2)4 increases with the length of the G-tract (n = 7 > n = 6 > n = 5 > n = 4 > n = 3). The CD spectra for d(GnT)4 in the presence of potassium exhibit positive peaks around 260 nm, consistent with the formation of parallel topologies. These peaks are retained in sodium-containing buffers, but when n = 4, 5, or 6, CD maxima are observed around 290 nm, suggesting that these sequences [especially d(G5T)4] have some antiparallel characteristics. d(G3T2)4 adopts a parallel conformation in the presence of both sodium and potassium, while all the other d(GnT2)4 complexes exhibit predominantly antiparallel features. The properties of these complexes are also affected by the rate of annealing, and faster rates favor parallel complexes.

CCA-adding enzymes build and repair the 3'-terminal CCA sequence of tRNA. These unusual RNA polymerases use either a ribonucleoprotein template (class I) or pure protein template (class II) to form mock base pairs with the Watson-Crick edges of incoming CTP and ATP. Guided by the class II Bacillus stearothermophilus CCA-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. We were able to transform the CCA-adding enzyme into a (U,G)-adding enzyme that incorporates UTP and GTP instead of CTP and ATP; we transformed the related Aquifex aeolicus CC- and A-adding enzymes into UU- and G-adding enzymes and Escherichia coli poly(A) polymerase into a poly(G) polymerase; and we transformed the B. stearothermophilus CCA-adding enzyme into a poly(C,A) polymerase by mutations in helix J that appear, based on the apoenzyme structure, to sterically limit addition to CCA. We also transformed the B. stearothermophilus CCA-adding enzyme into a dCdCdA-adding enzyme by mutating an arginine that interacts with the incoming ribose 2' hydroxyl. Most importantly, we found that mutations in helix J can affect the specificity of the nucleotide binding site some 20 A away, suggesting that the specificity of both class I and II enzymes may be dictated by an intricate network of hydrogen bonds involving the protein, incoming nucleotide, and 3' end of the tRNA. Collaboration between RNA and protein in the form of a ribonucleoprotein template may help to explain the evolutionary diversity of the nucleotidyltransferase family.

Background: Sufficient androgen receptor (AR) activity is crucial for normal male sexual differentiation. Here we report on two unrelated 46, XY patients suffering from undervirilization and genital malformations. Both patients had a short polyglycine (polyG) repeat of 10 residues and a relatively long polyglutamine (polyQ) repeat of 28 and 30 residues within the transactivation domain of the AR. In addition, they also harbor a rare A645D substitution., Objective: We made a set of AR expression plasmid constructs with varying polyQ and polyG tract sizes in context with or without the A645D substitution and analyzed their in vitro transactivation capacity in transfected CHO cells., Results: We found that a short polyG repeat downmodulated AR activity to approximately 60-65% of the wild-type receptor. This effect was aggravated by A645D in context of a long polyQ repeat to less than 50% activity. In contrast, in the context of a short polyQ and a short polyG repeat, the A645D mutation rescues AR activity to almost wild-type levels, demonstrating a contradictory effect of this mutation, depending on the size of the polymorphic repeats., Conclusions: A combination of a short polyG repeat with a long polyQ repeat and an A645D substitution might contribute to the development of virilization disorders and explain the observed phenotypes of our patients as a form of androgen insensitivity. The whole recreation of AR sequence variations including individual polymorphic repeat sizes could unravel possible interference of mutations and variations on AR activity by in vitro transfection.

Polymorphic RNA conformations may serve as potential targets for structure specific antiviral agents. As an initial step in the development of such drugs, the interaction of a wide variety of compounds which are characterized to bind to DNA through classical or partial intercalation or by mechanism of groove binding, with the A-form and the protonated form of poly(rC).poly(rG), been evaluated by multifaceted spectroscopic and viscometric techniques. Results of this study suggest that (i) ethidium intercalates to the A-form of RNA, but does not intercalate to the protonated form, (ii) methylene blue intercalates to the protonated form of the RNA but does not intercalate to the A-form, (iii) actinomycin D does not bind to either conformations of the RNA, and (iv) berberine binds to the protonated form by partial intercalation process, while its binding to the A-form is very weak. The DNA groove binder distamycin A has much higher affinity to the protonated form of the RNA compared to the A-form and binds to both structures by non-intercalative mechanism. We conclude that the binding affinity characteristics of these DNA binding molecules to the RNA conformations are vastly different and may serve as data for the development of RNA based antiviral drugs.

A guanine (G)-quadruplex DNA motif has recently emerged as a biologically important structure that is believed to interfere with telomere maintenance by telomerase. G-quadruplexes exhibit four-stranded structures containing one or more nucleic acid strands with central channel able to accommodate metal cations. Coordination of certain metal cations stabilizes G-quadruplex as with some promising small organic molecules that promote the formation and/or stabilization of G-quadruplex. Among many techniques employed to explore properties of G-quadruplexes, the fluorescence resonance energy transfer (FRET) technique has been recognized as a powerful tool to study G-quadruplex formation. This review summarizes the current developments in the uses of FRET technique for the fundamental structural investigations and its practical applications. Applications include FRET-based selection of efficient quadruplex-binding ligands, design of a nanomolecular machine, and a molecular aptamer beacon for protein recognition. We also describe a technique for detection of potassium ions in aqueous solution with the use of quadruplex-based sensor (potassium-sensing oligonucleotide).

Oligonucleotides (ODN) with hexameric motifs containing central unmethylated CpG dinucleotides are immunostimulatory. Also ODN with continuous guanosines (polyG motif) show a wide range of immunological activity. Depending on the position, the chemical property of the ODN backbone and the cell type, polyG motifs have either an enhancing or a suppressing effect on the immunostimulatory activity of the CpG-ODN. Microglial cells are central components of the innate immune system of the brain and are activated by CpG-ODN in vitro and in vivo. Here we present the analysis of the immunomodulatory effects of CpG-ODN carrying a polyG motif on the microglial cell line N9. Our data show that N9 cells express Toll-like receptor 9 (TLR9) and are activated by CpG-ODN, which leads to expression of interleukin-12p40 (IL12p40), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). A 3'-end polyG motif inhibits phosphothioate (PS) CpG-ODN immunostimulatory activity but enhances the immunostimulatory activity of phosphodiester (PE) CpG-ODN. Correspondingly, a 3'-end polyG motif improves the cellular uptake of PE CpG-ODN but does not change their cellular distribution pattern. Furthermore, PE CpG-ODN with a 3'-end polyG motif interact with a much higher number of cellular proteins than PE CpG-ODN. These data indicate that the 3'-end polyG motif could enhance the immunostimulatory activity of PE CpG-ODN in microglial N9 cells through increasing interaction with cellular proteins. Therefore PE CpG-ODN containing a 3'-end polyG motif resulting in increased immunostimulatory activity might be promising alternate analogues for studies in the central nervous system.

Synthetic immunostimulatory nucleic acids such as CpG DNA are being harnessed therapeutically as vaccine adjuvants, anticancer or antiallergic agents. Efforts to identify nucleic acid-based agents capable of more specifically modulating the immune system are being developed. The current study identifies a novel class of single-stranded oligoribonucleotides (ORN) containing unmethylated CpG motifs and a poly(G) run at the 3' end (CpG ORN) that directly stimulate human CD14+CD11c+ monocytes but not dendritic cells or B cells. CpG ORN activate NF-kappaB and p38 MAPK, resulting in IL-6 and IL-12 production and costimulatory molecule up-regulation but not IFNalpha. Methylation of cytosine at the 5' portion in core CpG motif abrogates such activation. TLR3, 7, 8, or 9 alone did not confer response to CpG ORN, in contrast to previously reported respective nucleic acid ligands. These data suggest that CpG ORN represent a novel class of synthetic immunostimulatory nucleic acids with distinct target cells, receptors, and functions from that of previously known immunomodulatory nucleic acids.

Background: The transcription factor c-Myb is expressed in hematopoietic progenitor cells and other rapidly proliferating tissues, regulating genes important for proliferation, differentiation and survival. The DNA-binding domain (DBD) of c-Myb contains three tandemly arranged imperfect repeats, designated Myb domain R1, R2 and R3. The three-dimensional structure of the DBD shows that only the second and third Myb domains are directly involved in sequence-specific DNA-binding, while the R1 repeat does not contact DNA and only marginally affects DNA-binding properties. No structural information is available on the N-terminal 30 residues. Since deletion of the N-terminal region including R1 plays an important role in oncogenic activation of c-Myb, we asked whether this region confers properties beyond DNA-binding to the neighbouring c-Myb DBD., Results: Analysis of a putative RNA-binding function of c-Myb DBD revealed that poly(G) preferentially inhibited c-Myb DNA-binding. A strong sequence-selectivity was observed when different RNA polymers were compared. Most interesting, the poly(G) sensitivity was significantly larger for a protein containing the N-terminus and the R1-repeat than for the minimal DNA-binding domain., Conclusion: Preferential inhibition of c-Myb DNA binding by poly(G) RNA suggests that c-Myb is able to interact with RNA in a sequence-selective manner. While R2 and R3, but not R1, are necessary for DNA-binding, R1 seems to have a distinct role in enhancing the RNA-sensitivity of c-Myb.

The Ki-ras gene is frequently mutated and/or overexpressed in human cancer. Since it is suspected to play a key role in the pathogenesis of many tumors, there is interest to search for strategies aiming at the specific inhibition of this oncogene. In this paper, we investigated the capacity of a 20 mer G-rich oligonucleotide (ODN20) conjugated to high molecular weight monomethoxy polyethylene glycol (MPEG) to inhibit the expression of the Ki-ras gene and the proliferation of pancreatic cancer cells. The conjugate, MPEG ODN20, was designed to form a triplex with a critical pur/pyr sequence located in the promoter of the Ki-ras gene. To make the conjugate resistant to endogenous and exogenous nucleases, five phosphorothioate linkages were introduced in its backbone. Confocal microscopy and FACS experiments showed that MPEG ODN20 had a higher capacity to penetrate the cell membranes and accumulate in the nucleus of Panc-1 cells than ODN20. Incubation of Panc-1 cells with MPEG ODN20 reduced specifically the levels of Ki-ras mRNA and RAS protein p21RAS. A single-dose administration of MPEG ODN20 was sufficient to inhibit cell proliferation by about 50% compared with control. By contrast, the antiproliferative activity of the unconjugated ODN20 analog was found to be not significant. Band-shift and footprinting experiments showed that MPEG ODN20 formed a weak triplex (Kd approximately 1.5 microM at 37 degrees C, 50 mM Tris-acetate, pH 7.4, 10 mM NaCl, 10 mM MgCl2, 5 mM spermidine) with the Ki-ras pyr/pur motif, suggesting that its bioactivity can hardly be mediated by a triplex-based mechanism. Here, we provide evidence that, in vitro, ODN20 and MPEG ODN20 competitively inhibit the binding to the Ki-ras pur/pyr motif of a nuclear protein, suggesting that the activity of MPEG ODN20 occurs with an aptameric mechanism. The biological implications of this study are discussed.

A ribonuclease, with a molecular mass of 23kDa, and much higher activity toward poly(U) than poly(C) and only negligible activity toward poly(A) and poly(G), was isolated from the aqueous extract of Chinese ginseng (Panax ginseng) flowers. The ribonuclease was unadsorbed on diethylaminoethyl-cellulose and adsorbed on Affi-gel blue gel and carboxymethyl-cellulose. High activity of the ribonuclease was maintained at pH 6-7. On either side of this pH range, there was a precipitous drop in enzyme activity. The activity of the enzyme peaked at 50 degrees C and fell to about 20% of the maximal activity when the temperature was lowered to 20 degrees C or raised to 80 degrees C. The characteristics of this ribonuclease were different from those of ribonuclease previously purified from ginseng roots.

Fragile X Mental Retardation protein (FMRP) is an RNA-binding protein that contains multiple domains with apparently differential affinity to mRNA and to the ribonucleotide homopolymer poly(G). Attempts have been made to map the RNA-binding sites along the protein sequence with a view to determining which of the KH1, KH2 and RGG domains are required to recognize and bind to RNA. While these studies have greatly contributed to the delineation of domains that bind homopolymers or mRNA in vitro, little is known concerning their implications in FMRP function(s) in vivo. To address this question, we have prepared a series of FMRP versions, in which each known in vitro functional domain has been individually deleted, leaving the rest of the protein intact. Constructs with deletions in the protein-protein interaction and RNA-binding as well as in the phosphorylation domains were expressed in STEK-KO cells lacking FMRP and their recruitment into polyribosomal mRNPs and their intra-cellular localization were determined. Our results indicate that the KH RNA-binding domains and the Protein-Protein Interacting domain are essential for FMRP to associate with polyribosomal mRNPs, while the RGG box and the phosphorylated domains are dispensable.

The Schizosaccharomyces pombe Ku70-Ku80 heterodimer is required for telomere length regulation. Lack of pku70+ results in telomere shortening and striking rearrangements of telomere-associated sequences. We found that the rearrangements of telomere-associated sequences in pku80+ mutants are Rhp51 dependent, but not Rad50 dependent. Rhp51 bound to telomere ends when the Ku heterodimer was not present at telomere ends. We also found that the single-stranded G-rich tails increased in S phase in wild-type strains, while deletion of pku70+ increased the single-stranded overhang in both G2 and S phase. Based on these observations, we propose that Rhp51 binds to the G-rich overhang and promotes homologous pairing between two different telomere ends in the absence of Ku heterodimer. Moreover, pku80 rhp51 double mutants showed a significantly reduced telomere hybridization signal. Our results suggest that, although Ku heterodimer sequesters Rhp51 from telomere ends to inhibit homologous recombination activity, Rhp51 plays important roles for the maintenance of telomere ends in the absence of the Ku heterodimer.

The hydroxyl radical generated by UV irradiation of hydrogen peroxide cause an extensive damage to guanine residues of ribohomopolymer, polyguanylic acid, poly (G) as investigated by spectrophotometric measurements, agarose gel electrophoresis, Sephadex G-200 gel filtration and DEAE Sephadex A-25 column chromatography. Native and ROS-poly (G) were highly immunogenic inducing high titre antibodies in rabbits. The antibodies showed wide range of cross reactivity with various synthetic polynucleotides exhibiting B-, A-, and allied conformations. The diverse antigen binding characteristics of the induced antibodies resembles to those of naturally occurring lupus anti-DNA autoantibodies. Sera from various SLE patients showed preferential binding to ROS-poly (G) than native poly (G), indicating that oxidatively modified guanine residues are better recognised. The significance of these findings in the induction of SLE anti-DNA autoantibodies by oxygen free radicals modified guanine residues in DNA has been discussed.

A ribonuclease, with a molecular mass of 13 kDa and a ubiquitin-like N-terminal sequence, has been isolated from fruiting bodies of the mushroom Termitomyces globulus. The ribonuclease demonstrated ribonucleolytic activity toward poly A, poly C, poly G and poly U, with the activity toward poly A and poly C being much higher than that toward poly G and poly U. The ribonuclease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-Sepharose. The enzyme required a temperature of 70 degrees C for expression of maximal activity. However, the enzyme expressed nearly the same optimal activity over a wide pH range of 5.0-8.0.

Trypanosomes, protozoan parasites from the order Kinetoplastida, have to deal with environmental changes during the interaction with their hosts. Trypanosoma cruzi, the causative agent of Chagas' disease, uses post-transcriptional mechanisms to regulate gene expression. However, few RNA-binding proteins involved in mRNA turnover control have been identified to date. In this work, an RNA recognition motif (RRM)-type RNA-binding protein family named T. cruzi RNA-binding protein (TcRBP) and composed of at least six members was identified. The genomic organization of four members revealed a head to tail arrangement within a region of 15 kilobase pairs. TcRBP members have a common RRM and different auxiliary domains with a high content of glycine, glutamine, and histidine residues within their N- and C-terminal regions. TcRBPs differ in their expression patterns as well as in their homoribopolymer binding interaction in vitro, although they preferentially recognize poly(U) and poly(G) RNAs. An interesting observation was the relaxed RNA-binding interactions with several trypanosome transcripts in vitro. In contrast, co-immunoprecipitation experiments of TcRBP-containing ribonucleoprotein complexes formed in vivo revealed a highly restricted binding interaction with specific RNAs. Several TcRBP-containing complexes are stage-specific and, in some cases, bear the poly(A)-binding protein TcPABP1. Altogether, these results suggest that TcRBPs might be modulated in vivo, to favor or preclude the interaction with specific transcripts in a developmentally regulated manner.

Methotrexate (MTX), an antineoplastic agent, demonstrates phototoxicity. The mechanism of damage to biomacromolecules induced by photoirradiated MTX was examined using 32P-labeled DNA fragments obtained from a human gene. Photoirradiated MTX caused DNA cleavage specifically at the underlined G in 5'-GG and 5'-GGG sequences in double-stranded DNA only when the DNA fragments were treated with piperidine, which suggests that DNA cleavage was caused by base modification with little or no strand breakage. With denatured single-stranded DNA the damage occurred at most guanine residues. The amount of formation of 8-hydroxy-2'-deoxyguanosine (8-oxodGuo), an oxidative product of 2'-deoxyguanosine, in double-stranded DNA exceeded that in single-stranded DNA. These results suggest that photoirradiated MTX participates in 8-oxodGuo formation at the underlined G in 5'-GG and 5'-GGG sequences in double-stranded DNA through electron transfer, and then 8-oxodGuo undergoes further oxidation into piperidine-labile products. Fluorescence measurement, high-pressure liquid chromatography and mass spectrometry have demonstrated that photoexcited MTX is hydrolyzed into 2,4-diamino-6-(hydroxymethyl)pteridine (DHP). DNA damage induced by DHP was observed in a similar manner as was the damage induced by MTX. The extent of DNA damage and the formation of 8-oxodGuo by DHP were much larger than those induced by MTX. The kinetic analysis, based on the time course of DNA oxidation by photoirradiated MTX, suggests that DNA damage is caused by photoexcited DHP rather than by photoexcited MTX. In conclusion, photoexcited MTX undergoes hydrolysis through intramolecular electron transfer, resulting in the formation of DHP, which exhibits a phototoxic effect caused by oxidation of biomacromolecules through photoinduced electron transfer.

Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-DNA) triggers innate immune cells through the pattern recognition receptor Toll-like receptor 9 (TLR-9). CpG-DNA possesses potent immunostimulatory effects on macrophages, dendritic cells, and B lymphocytes. Therefore, CpG-DNA contributes to inflammation during the course of bacterial infections. In contrast to other TLR-dependent microbial patterns, CpG-DNA is a strong inductor of IL-12. Thus, it acts as a Th1-polarizing agent that can be utilized as potent vaccine adjuvant. To assess the role of CpG-DNA in immune reactions in the CNS, we analyzed the effects of CpG-DNA on microglial cells in vitro and in vivo. Primary microglial cells as well as microglial cell lines express TLR-9 mRNA. Consequently, CpG-DNA activated microglial cells in vitro and induced TNF-alpha, IL-12p40, IL-12p70, and NO. Furthermore, MHC class II, B7-1, B7-2, and CD40 molecules were up-regulated. In addition, phagocytic activity of microglia was enhanced. After intracerebroventricular injection of CpG-DNA, microglial cells were activated and produced TNF-alpha and IL-12p40 transcripts, as shown by in situ hybridization. These results indicate that microglia is sensitive to CpG-DNA. Thus, bacterial DNA containing CpG motifs could not only play an important role during infections of the CNS, but also might trigger and sustain Th1-dominated immunopathogenic reactions.

The loss of the fragile X RNA binding protein, FMRP, causes macroorchidism and mental retardation in man. The discovery of a mouse ortholog led to the development of several FMRP knockout mouse strains that recapitulate some features of the disease. As mouse and human FMRPs differ in several amino acids in their RNA binding domains, we compared the RNA binding profiles of these two orthologs. Five variant FMRPs, whose differences arose from alternative splicing and mutation within the conserved RNA binding domains, were examined. Homoribopolymer binding studies showed that human FMRPs (hFMRP) bound a broader range of single-stranded mimetics than mouse FMRPs (mFMRP) and these interactions were both complex and cooperative. hFMRP and mFMRP also displayed significant preferences toward binding their own mRNA; specifically we found that the mFMRP isoforms bind mFMR1 mRNA much more tightly than their human counterparts. Finally, these data demonstrate that each FMRP variant binds RNAs uniquely, resulting in a set of proteins with differing affinities., ((c)2002 Elsevier Science (USA).)

The TLS/FUS gene is involved in a recurrent chromosomal translocation in human myxoid liposarcomas. We previously reported that TLS is a potential splicing regulator able to modulate the 5'-splice site selection in an E1A pre-mRNA. Using an in vitro selection procedure, we investigated whether TLS exhibits a specificity with regard to RNA recognition. The RNAs selected by TLS share a common GGUG motif. Mutation of a G or U residue within this motif abolishes the interaction of TLS with the selected RNAs. We showed that TLS can bind GGUG-containing RNAs with a 250 nm affinity. By UV cross-linking/competition and immunoprecipitation experiments, we demonstrated that TLS recognizes a GGUG-containing RNA in nuclear extracts. Each one of the RNA binding domains (the three RGG boxes and the RNA recognition motif) contributes to the specificity of the TLS.RNA interaction, whereas only RRM and RGG2-3 participate to the E1A alternative splicing in vivo. The specificity of the TLS.RNA interaction was also observed using as natural pre-mRNA, the G-rich IVSB7 intron of the beta-tropomyosin pre-mRNA. Moreover, we determined that RNA binding specificities of TLS and high nuclear ribonucleoprotein A1 were different. Hence, our results help define the role of the specific interaction of TLS with RNA during the splicing process of a pre-mRNA.

Using a model system of young, senescent and SV40-immortalized WI-38 fibroblasts, we identified two mRNAs upregulated in immortalized cells (imup-1, immortalization-upregulated protein 1, and imup-2). Compared to normal tissues, both genes were more frequently expressed in cancer cells. The open reading frame of imup-1 spans 321bp, coding for a 10.9 kDa protein of 106 amino acids, while an insertion of 59bp in the otherwise identical mRNA of imup-2 leads to a frameshift, resulting in an 8.5 kDa protein of 85 amino acids. Database searches identified these genes on chromosome 19, which could account for the cloned imup-1 and imup-2 transcripts by alternative splicing. Southern blot analysis of digested genomic DNA confirmed that both transcripts are derived from a single locus. The expressed proteins IMUP-1 and IMUP-2 share 46 identical N-terminal amino acids, whereas the C-termini are unrelated. Green fluorescent protein-fusions of both IMUP-1 and IMUP-2 accumulated in the nucleus of HeLa cells. The C-terminus of IMUP-1 contains a bipartite nuclear localization signal, the deletion of which impaired nuclear translocation. In-vitro translated proteins bound to poly(rG), but did not interact with single-stranded DNA or double-stranded DNA. The nuclear localization of IMUP-1 and IMUP-2 as well as the upregulation of both underlying mRNAs in immortalized cells suggest a function in immortalization.

The RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene has been expressed as a nonfusion protein in bacterial cells and purified to homogeneity using sequential chromatographic columns. The purified NS5B protein exhibited RNA-dependent RNA polymerase activity using poly(A) template and the K(m) and V(max) were determined as 8.4 microM and 1976 pmol/mg-min, respectively. This full-length NS5B protein exhibited much stronger binding affinity toward the 30-mer poly(G) than other homopolymeric RNAs of the same size. For the first time, we demonstrate that the HCV NS5B was able to bind various ribonucleotides. Using a panel of oligonucleotides varying in length, we studied the NS5B catalytic efficiency and proposed the size of the NS5B active site to be 8-10 nucleotides. The multifunctional nature of NS5B protein is also discussed and compared with other viral RNA polymerases., (Copyright 2000 Academic Press.)

We have shown that muramyl dipeptide (MDP) conjugated to a 10-mer polyguanylic acid (PolyG) is specifically internalized by macrophages through scavenger receptor (SCR)-mediated endocytosis. Macrophages activated by PolyG-MDP displayed about 20-fold higher cytotoxic activity against nonmacrophage tumor cells compared to that elicited by free MDP. The PolyG-MDP was found to trigger the secretion of higher levels of interleukin-6, interleukin-1alpha, TNF-alpha, and nitric oxide in comparison to free MDP. Addition of antibodies directed against IL-6 and TNF-alpha to macrophage culture completely abrogated the tumoricidal response of PolyG-MDP, indicating that these two cytokines are primarily responsible for bioefficacy. This general approach of PolyG as a vehicle may find wide application in the delivery of genes and antisense oligonucleotides to macrophages., (Copyright 2000 Academic Press.)

Aminoacyl-tRNA synthetases of higher eukaryotes possess polypeptide extensions in contrast to their prokaryotic counterparts. These extra domains of poorly understood function are believed to be involved in protein-protein or protein-RNA interactions. Here we showed by gel retardation and filter binding experiments that the repeated units that build the linker region of the bifunctional glutamyl-prolyl-tRNA synthetase had a general RNA-binding capacity. The solution structure of one of these repeated motifs was also solved by NMR spectroscopy. One repeat is built around an antiparallel coiled-coil. Strikingly, the conserved lysine and arginine residues form a basic patch on one side of the structure, presenting a suitable docking surface for nucleic acids. Therefore, this repeated motif may represent a novel type of general RNA-binding domain appended to eukaryotic aminoacyl-tRNA synthetases to serve as a cis-acting tRNA-binding cofactor.

Molecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5' and 3' untranslated regions. While it is well-known that chloroplast mRNAs are unstable in the absence of certain nucleus-encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild-type cells. Here we used a poly(G)18 sequence, which impedes both 5'-->3' and 3'-->5' exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild-type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus-encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions -20, +25, +165 or +25/+165 relative to the mature petD 5' end, mRNAs accumulate with 5' ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5' end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild-type cells by a 5'-->3' exoribonucleolytic activity. In the case of the -20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5' end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5' ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild-type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) -20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.

The RNase activity of barnase mutants obtained by the permutation of modules or secondary structure units was investigated. Four of the 45 mutants had weak but distinct RNase activity, and they had unique optimum pHs and temperatures like natural enzymes. One of the active mutants had an ordered conformation, but the others did not. An active mutant having disordered conformation formed an ordered conformation in the presence of GMP, which is an inhibitor of this mutant. These results indicate that the amino acid sequences derived from barnase have sufficient plasticity to be rearranged into different proteins with basal enzymatic properties.

In the fission yeast Schizosaccharomyces pombe the nrd1(+) gene encoding an RNA binding protein negatively regulates the onset of differentiation. Its biological role is to block differentiation by repressing a subset of the Ste11-regulated genes essential for conjugation and meiosis until the cells reach a critical level of nutrient starvation. By using the phenotypic suppression of the S. pombe temperature-sensitive pat1 mutant that commits lethal haploid meiosis at the restrictive temperature, we have cloned ROD1, a functional homologue of nrd1(+), from rat and human cDNA libraries. Like nrd1(+), ROD1 encodes a protein with four repeats of typical RNA binding domains, though its amino acid homology to Nrd1 is limited. When expressed in the fission yeast, ROD1 behaves in a way that is functionally similar to nrd1(+), being able to repress Ste11-regulated genes and to inhibit conjugation upon overexpression. ROD1 is predominantly expressed in hematopoietic cells or organs of adult and embryonic rat. Like nrd1(+) for fission yeast differentiation, overexpressed ROD1 effectively blocks both 12-O-tetradecanoyl phorbol-13-acetate-induced megakaryocytic and sodium butyrate-induced erythroid differentiation of the K562 human leukemia cells without affecting their proliferative ability. These results suggest a role for ROD1 in differentiation control in mammalian cells. We discuss the possibility that a differentiation control system found in the fission yeast might well be conserved in more complex organisms, including mammals.

Polyadenylation of mRNA has been shown to target the RNA molecule for rapid exonucleolytic degradation in bacteria. To elucidate the molecular mechanism governing this effect, we determined whether the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated PNPase degraded polyadenylated and non-polyadenylated RNAs at similar rates. However, when the two molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. The same phenomenon was observed with polyuridinylated RNA. The poly(A) tail has to be located at the 3' end of the RNA, as the addition of several other nucleotides at the 3' end prevented competition for polyadenylated RNA. In RNA-binding experiments, E. coli PNPase bound to poly(A) and poly(U) sequences with much higher affinity than to poly(C) and poly(G). This high binding affinity defines poly(A) and poly(U) RNAs as preferential substrates for this enzyme. The high affinity of PNPase for polyadenylated RNA molecules may be part of the molecular mechanism by which polyadenylated RNA is preferentially degraded in bacterial cells.

Both cis- and trans-RuCl2(DMSO)4 (cis-Ru and trans-Ru) react with ApG, GpA, d(ApG) and d(GpA) to yield products with bifunctional metal coordination of the bases. For each dinucleotide one major product and several minor species are formed. This is in contrast to previous results on analogous reactions between trans-Ru and d(GpG) where a substantial amount of an intermediate species was found. The rates of reaction between dinucleotides and cis-Ru are approximately 20-fold slower than for trans-Ru. The compounds formed with the two isomers exhibit identical proton NMR spectra, suggesting the same coordination mode for ruthenium in the final product. The two purine bases are coordinated to ruthenium through N7 in a head-to-head conformation with the glycosidic angles being in the anti range. Coupling constants indicate a relatively pure 3'-endo conformation for the 5'-sugar and mainly 2'-endo for the 3'-sugar. The similar bifunctional binding mode of cis- and trans-Ru(II) with dinucleotides as evident from the NMR spectra are in contrast to the different mode of interaction suggested earlier for cis- and trans-Ru complexes with DNA. trans-Ru interacts with the deoxyoctanucleotide d(CCTGGTCC), giving two main products during the first 2 h of incubation time. Four H8 guanine resonances are shifted downfield, characteristic of N7 metal coordination. The products are not analyzed in detail, but it is suggested that the structures may be described as two chiral G(N7/N7) chelates.

The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin beta to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin alpha/beta or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos.

The effect of the hydroxyl radical on polyguanylic acid [poly(G)] was investigated with regard to progressive increase of autoantibodies against it in systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS). Rabbits immunized with both native and ROS-poly(G) induced high titre antibodies. Immune IgG exhibited a high degree of specificity towards the immunogen, reiterated visually by a gel retardation assay. The induced antibodies showed a wide range of cross-reactivity with various synthetic polynucleotides exhibiting B-, A-, and allied conformations. The specificity of induced antibodies resembled the diverse binding characteristics of lupus anti-DNA autoantibodies. Moreover, sera from scleroderma patients showed binding to native and ROS-poly(G). SLE and PSS autoantibodies showed preferential recognition of ROS-poly(G) over native poly(G). These results demonstrate that the hydroxyl modified guanine residues in DNA and RNA can induce circulating SLE and PSS autoantibodies., (Copyright 1998 Academic Press)