Bent oligonucleotide duplexes as HMGB1 inhibitors: a comparative study

In this work we explore the ability of a chimeric LNA/DNA bent duplex, in which the kink is induced by 2 unpaired adenines in the middle of one strand, to bind HMGB1, a protein involved in many inflammatory processes. The LNA/DNA duplex was compared with the corresponding full DNA and PNA/DNA chimera duplexes from a thermodynamic and spectroscopic point of view.     

Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

The condensation of nucleic acids into well-defined particles is an integral part of several approaches to artificial cellular delivery. Improvements in the efficiency of nucleic acid delivery in vivo are important for the development of DNA- and RNA-based therapeutics. Presently, most efforts to improve the condensation and delivery of nucleic acids have focused on the synthesis of novel condensing agents. However, short oligonucleotides are not as easy to condense into well-defined particles as gene-length DNA polymers and present particular challenges for discrete particle formation. We describe a novel strategy for improving the condensation and packaging of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. 2 kb). These non-covalent assemblies possess single-stranded nicks or single-stranded gaps at regular intervals along the duplex backbones. The condensation behavior of nicked- and gapped-DNA duplexes was investigated using several cationic condensing agents. Transmission electron microscopy and light-scattering studies reveal that these DNA duplexes condense much more readily than short duplex oligonucleotides (i.e. 21 bp), and more easily than a 3 kb plasmid DNA. The polymeric condensing agents, poly-l-lysine and polyethylenimine, form condensates with nicked- and gapped-DNA that are significantly smaller than condensates formed by the 3 kb plasmid DNA. These results demonstrate the ability for DNA structure and topology to alter nucleic acid condensation and suggest the potential for the use of this form of DNA in the design of vectors for oligonucleotide and gene delivery. The results presented here also provide new insights into the role of DNA flexibility in condensate formation.     

Improved selectivity for the binding of naphthyridine dimer to guanine-guanine mismatch.

Naphthyridine dimer composed of two 2-amino-1,8-naphthyridines and a connecting linker strongly binds to guanine-guanine (G-G) mismatch in duplex DNA. In order to improve G-G selectivity for the binding, we have examined structure modification of the linker. A new naphthyridine dimer possessing 3,6-diazaoctanedioic acid linker binds to G-G mismatch with an association constant of 1.18 x 10(7) M(-1), which is somewhat weaker than that of the original naphthyridine dimer having a shorter connecting linker. However, the binding of the modified naphthyridine dimer to G-A mismatch was almost negligible as compared to that of the original. This results in a net increase of the selectivity for the binding to G-G mismatch by 4-folds.     

Spongelike alginate nanoparticles as a new potential system for the delivery of antisense oligonucleotides.

The aim of this study was to design a new antisense oligonucleotide (ON) carrier system based on alginate nanoparticles and to investigate its ability to protect ON from degradation in the presence of serum. Pharmacokinetics and tissue distribution of ON-loaded nanoparticles have been determined after intravenous administration. An original and dynamic process for ON loading into polymeric nanoparticles has been applied. It is based on the diffusion of ON or ON/polylysine complex into the nanoparticle or the alginate gel, respectively. Indeed, the single coincubation of ON with nanoparticles led, within a few days, to an extremely efficient association. The diffusion kinetic of ON was shown to be dependent on several parameters, incubation temperature, ON concentration, presence or absence of polylysine, polylysine molecular weight, and nanoparticle preparation procedure. This new alginate-based system was found to be able to protect [33P]-radiolabeled ON from degradation in bovine serum medium and to modify their biodistribution, as an important accumulation of radioactivity was observed in the lungs, in the liver, and in the spleen after intravenous administration into mice. ON may be associated efficiently with calcium alginate in a colloidal state. Such nanosponges are promising carriers for specific delivery of ON to lungs, liver, and spleen.     

Probing structural factors stabilizing antisense oligonucleotide duplexes: NMR studies of a DNA.DNA duplex containing a formacetal linkage.

The duplex formed by annealing the formacetal backbone modified dodecamer d-(CGCGTTOCH2OTTGCGC) to its complementary strand, d(GCGCAAAACGCG) (duplex I), has been studied by NMR techniques and analyzed with reference to its unmodified counterpart (duplex II). Comparison of parameters such as 2D cross-peak intensities, coupling constants, and spectral patterns indicates that structural perturbations caused by the incorporation of the formacetal linkage are minimal and localized to the central T4.A4 block. Duplex I adopts a B-type helical conformation with regular Watson-Crick base pairing and normal minor groove width. The methylene group is accommodated along the phosphate backbone in a conformation similar to that of the PO2 group found in the B-form DNA family. The central T6-T7 base pairs of duplex I melt simultaneously with the duplex, indicating a cooperative transition to single strands. Although the formacetal linkage affects global melting, as evidenced by a 3 degree C reduction in Tm for duplex I with respect to duplex II, the present study indicates that this is not the result of localized premelting at the formacetal site of duplex I but rather reflects the subtle interplay of several structural and energy factors which need to be further explored.     

Drosophila: a "model" model system to study neurodegeneration

The fruit fly, Drosophila melanogaster, is a powerful model genetic organism that has been used since the turn of the previous century in the study of complex biological problems. In the last decade, numerous researchers have focused their attention on understanding neurodegenerative diseases by utilizing this model system. Numerous Drosophila mutants have been isolated that profoundly affect neural viability and integrity of the nervous system with age. Additionally, many transgenic strains have been developed as models of human disease conditions. We review the existing Drosophila neurodegenerative mutants and transgenic disease models, and discuss the role of the fruit fly in therapeutic development for neurodegenerative diseases.     

Raman study of the interaction between polyamines and a GC oligonucleotide.

The interaction between the oligonucleotide d[G(CG)(7)]. d[C(GC)(7)] and the three biogenic polyamines putrescine, spermidine, and spermine under physiological conditions has been studied by Raman spectroscopy. The results indicate the formation of highly ordered aggregated structures in solution, largely stabilized by electrostatic attractions, which have been described as cholesteric phases. Aggregation seems to be preceded by a partial B --> Z conformational transition for spermidine and spermine, which would allow for a deeper oligonucleotide-polyamine interaction. Interaction with the nucleic bases has also been evidenced for aggregates. At low polyamine concentrations the preferential binding sites are similar to those proposed for their interactions with ct-DNA. With increasing the polyamine concentration, the oligonucleotide-polyamine interactions involve both minor and major grooves, which is consistent with the formation of cholesteric phases.     

Drosophila melanogaster as a model system for the evaluation of anti-aging compounds

Understanding the causes of aging is a complex problem due to the multiple factors that influence aging, which include genetics, environment, metabolism and reproduction, among others. These multiple factors create logistical difficulties in the evaluation of anti-aging agents. There is a need for good model systems to evaluate potential anti-aging compounds. The model systems used should represent the complexities of aging in humans, so that the findings may be extrapolated to human studies, but they should also present an opportunity to minimize the variables so that the experimental results can be accurately interpreted. In addition to positively affecting lifespan, the impact of the compound on the physiologic confounders of aging, including fecundity and the health span-the period of life where an organism is generally healthy and free from serious or chronic illness-of the model organism needs to be evaluated. Fecundity is considered a major confounder of aging in fruit flies. It is well established that female flies that are exposed to toxic substances typically reduce their dietary intake and their reproductive output and display an artifactual lifespan extension. As a result, drugs that achieve longevity benefits by reducing fecundity as a result of diminished food intake are probably not useful candidates for eventual treatment of aging in humans and should be eliminated during the screening process.     

Ladybirds as a model system for the study of male-killing symbionts

Maternally inherited bacteria that kill male but not female hosts during embryogenesis occur in a number of aphidophagous coccinellids. Work on EnglishAdalia bipunctata (L.), has shown the causative agent of male-killing to be a member of the bacterial genusRickettsia. In coccinellids, the primary advantage of male-killing behaviour to the bacterium has been identified. Following male death, resource reallocation occurs through sibling egg cannibalism: female neonate larvae of infected mothers gain a significant survival advantage by eating the soma of their dead male siblings. In addition, daughters of infected females suffer a reduced risk of cannibalism as a result of the lower egg hatch rate in infected clutches. Predictions as to which species of coccinellid are liable to harbour male-killers may be made on the basis of the selective advantages of male-killing identified inA. bipunctata. Species which may harbour male-killers are likely to lay eggs in clutches, show sibling egg cannibalism, and exhibit high neonate mortality. Recent work has shown male-killing to occur in a number of other aphidophagous coccinellids with the predicted characteristics. Molecular genetic analysis has putatively identified three bacterial symbionts associated with male-killing, coming from three phylogenetically distant bacterial taxa. We therefore suggest that within coccinellids that possess these features, male-killing may evolve in a taxonomically diverse range of inherited bacteria. The implications of the presence of male-killing bacteria on the population demography of host coccinellids, and on host mitochondrial DNA variability are discussed. The aphidophagous coccinellids are proposed as a model system for studying the evolution and consequences of infection with male-killers.     

N-Acetyl-D-glucosamine binding lectins. A model system for the study of binding specificity

Synthetic glycoconjugates prepared by the direct reductive amination of di-N-acetylchitobiose and tetra-N-acetyl-chitotetraose to poly-l-lysine with sodium cyanoborohydride have been used to explore the binding specificities of the lectins wheat germ agglutinin and Bandeiraea simplicifolia II. These conjugates are effective precipitating antigens for these lectins, and hapten inhibition experiments, employing the per-N-acetylated oligomers of chitin as inhibitors, demonstrate that wheat germ agglutinin and Bandeiraea simplicifolia II lectin have binding sites complementary to three and two contiguous β 1,4-linked N-acetyl-d-glucosamine residues, respectively, in agreement with conclusions reached using other methods. Conjugates prepared by this technique should be useful for examining the binding specificities of other lectins, and the results of a study of the effect of chain length of the hapten on the affinity of the lectin for these conjugates should provide guidance in selection of the hapten most appropriate for these studies.     

The zebrafish as a model system for assessing the reinforcing properties of drugs of abuse

Recent reports make use of the zebrafish to study complex behavior such as addiction, anxiety, or learning and memory. We have established reliable tests and appropriate controls to measure these behavioral parameters in the zebrafish adult. Our assays are robust enough to permit the detection of dominant mutations affecting drug-induced reward, and therefore can be used in forward genetic screens. We provide the reader with the technical details of these tests, as well as their appropriate and crucial, although often overlooked, control assays. In particular, our results make it possible to use the zebrafish as a promising model to identify new genetic components of the reward pathway, or other measurable behaviors.     

The fish heart as a model system for the study of myoglobin

A model is presented for myoglobin study based upon naturally occurring differences in myocardial myoglobin content in fish. The sea raven (Hemitripterus americanus) and the ocean pout (Macrozoarces americanus) have heart myoglobin contents of approx. 65 and 5 nmol/g wet wt, respectively. The maximal activities of enzymes associated with energy metabolism are similar in the two hearts. Isolated perfused hearts performed with similar efficiencies based upon similar rates of work, oxygen consumption and lactate production. Under normoxic perfusion conditions both hearts met 98% of the ATP demand by oxidative mechanisms. Myoglobin-rich sea raven hearts performed significantly better than myoglobin-poor ocean pout hearts under conditions of hypoxia and glycolytic blockage. The performance of sea raven hearts was impaired during hypoxia by decreasing the content of functional myoglobin with hydroxylamine. No effect upon performance was observed with the ocean pout heart. The data provide the first evidence that myoglobin plays a role in the maintenance of contractility in heart under hypoxic conditions.     

Electrophorus electricus as a model system for the study of membrane excitability

The stunning sensations produced by electric fish, particularly the electric eel, Electrophorus electricus, have fascinated scientists for centuries. Within the last 50 years, however, electric cells of Electrophorus have provided a unique model system that is both specialized and appropriate for the study of excitable cell membrane electrophysiology and biochemistry. Electric tissue generates whole animal electrical discharges by means of membrane potentials that are remarkably similar to those of mammalian neurons, myocytes and secretory cells. Electrocytes express ion channels, ATPases and signal transduction proteins common to these other excitable cells. Action potentials of electrocytes represent the specialized end function of electric tissue whereas other excitable cells use membrane potential changes to trigger sophisticated cellular processes, such as myofilament cross-bridging for contraction, or exocytosis for secretion. Because electric tissue lacks these functions and the proteins associated with them, it provides a highly specialized membrane model system. This review examines the basic mechanisms involved in the generation of the electrical discharge of the electric eel and the membrane proteins involved. The valuable contributions that electric tissue continues to make toward the understanding of excitable cell physiology and biochemistry are summarized, particularly those studies using electrocytes as a model system for the study of the regulation of membrane excitability by second messengers and signal transduction pathways.     

Evaluating the potential of fluorinated tyrosines as spectroscopic probes of local protein environments: a UV resonance Raman study

Ultraviolet resonance Raman (UVRR) studies designed to test the utility of fluorinated tyrosines as spectroscopic probes of the local environment are presented. Specifically, resonance Raman spectra of 2-fluoro-L-tyrosine and 3-fluoro-L-tyrosine (3-Y(f)) obtained with 229 nm excitation are reported. In contrast to the modest environmental dependence of the tyrosine resonance Raman spectrum, the spectrum of 3-Y(f) is found to be extremely dependent on the hydrogen bonding strength of the surrounding environment. Preliminary ab initio studies suggest that this behavior is due to normal modes having dominant contributions from the C-OH and C-F internal coordinates. Hydrogen bonding to the solvent perturbs the internal coordinate energetics and/or couplings, thereby altering the character of the normal modes and the corresponding transition frequencies and/or intensities. In addition to the solvent studies, 3-Y(f) is site specifically incorporated into the influenza hemagglutinin (HA) 100-107 peptide which binds to the Fv fragment of the 17/9 anti-HA(98-108) peptide antibody. These studies demonstrate that the spectrum of 3-Y(f) can be monitored in the presence of native tyrosine. In summary, the studies presented here demonstrate that 3-Y(f) holds exceptional promise as a probe of the protein environment.     

Oligonucleotide conjugates as potential antisense drugs with improved uptake,biodistribution, targeted delivery,and mechanism of action

This review summarizes the effect of conjugating small molecules and large biomacromolecules to antisense oligonucleotides to improve their therapeutic potential. In many cases, favorable changes in pharmacokinetic and pharmacodynamic properties were observed. Opportunities exist to change the terminating mechanism of antisense action or to enhance the RNase H mode of action via conjugate formation.     

Making cool drugs hot: isothermal titration calorimetry as a tool to study binding energetics

Characterization of the thermodynamics of binding interactions is important in improving our understanding of bimolecular recognition and forms an essential part of the rational drug design process. Isothermal titration calorimetry (ITC) is rapidly becoming established as the method of choice for undertaking such studies. The power of ITC lies in its unique ability to measure binding reactions by the detection of the heat change during the binding interaction. Since heat changes occur during many physicochemical processes, ITC has a broad application, ranging from chemical and biochemical binding studies to more complex processes involving enthalpy changes, such as enzyme kinetics. Several features of ITC have facilitated its preferential use compared to other techniques that estimate affinity. It is a sensitive, rapid, and direct method with no requirement for chemical modification or immobilization. It is the only technique that directly measures enthalpy of binding and so eliminates the need for van't Hoff analysis, which can be time consuming and prone to uncertainty in parameter values. Although ITC has facilitated the measurement of the thermodynamics governing binding reactions, interpretation of these parameters in structural terms is still a major challenge.     

Oncogene-transformed granulosa cells as a model system for the study of steroidogenic processes

Highly steroidogenic granulosa cell lines were established by transfection of primary granulosa cells from preovulatory follicles with SV40 DNA and Ha-ras oncogene. Progesterone production in these cells was enhanced to levels comparable to normal steroidogenic cells, by prolonged (> 12 h) stimulation with 8-Br-cAMP, forskolin and cholera toxin, which elevate intracellular cAMP. The steroidogenic capacity of individual lines correlated with the expression of the ras oncogene product (p21) and the morphology of the cells. Formation of the steroid hormones was associated with de novo synthesis of the mitochondrial cytochrome P450scc system proteins. Since cholesterol import into mitochondria is essential for steroidogenesis, the expression of the peripheral benzodiazepine receptor (PBR) and the sterol carrier protein 2 was characterized in these cells. The induction of the expression of the genes coding for both proteins appeared to be mediated, at least in part, by cAMP. Stimulation of the PBR by specific agonists enhanced progesterone production in these cells. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) dramatically suppressed the cAMP-induced steroidogenesis, in spite of enhanced intracellular cAMP levels, suggesting that TPA can modify the effects of cAMP. cAMP stimulation suppressed growth of transformed cells concomitantly with induction of steroidogenesis. The transformed cells lacked receptors for the native stimulants, the gonadotropic hormones. After transfection of the cells with a lutropin (LH) receptor expression plasmid, the LH and hCG response was reconstituted. In these newly established cell lines gonadotropins were able to stimulate the formation of cAMP and progesterone in a dose-dependent manner with an ED₅₀ characteristic of the native receptor. High doses caused desensitization to gonadotropins as observed in normal cells. These newly established oncogene-transformed granulosa cell lines can serve as a useful model to study inducible steroidogenesis and the effect of oncogene expression on this process.     

Mcg light chain dimer as a model system for ligand design: a docking study

Mcg light chain dimer has been extensively studied by crystallography and peptide binding studies to investigate its peptide cross-reactivity as well as to use it as a model system for designing space filling peptide ligands. Here we extend these investigations by utilizing automated docking. Mcg light chain dimer is an ideal model system for such study due to the availability of experimental data for both the native structure and the 14 complexes with various peptide ligands. We show the ability of the docking approach to reproduce the experimental structures and discuss the limitations associated with such outcomes. We demonstrate the usefulness of the docking approach in generating structural information otherwise not available from the experiment.     

Thermodynamics of RNA-RNA duplexes with 2- or 4-thiouridines: implications for antisense design and targeting a group I intron

Antisense compounds are designed to optimize selective hybridization of an exogenous oligonucleotide to a cellular target. Typically, Watson-Crick base pairing between the antisense compound and target provides the key recognition element. Uridine (U), however, not only stably base pairs with adenosine (A) but also with guanosine (G), thus reducing specificity. Studies of duplex formation by oligonucleotides with either an internal or a terminal 2- or 4-thiouridine (s(2)U or s(4)U) show that s(2)U can increase the stability of base pairing with A more than with G, while s(4)U can increase the stability of base pairing with G more than with A. The latter may be useful when binding can be enhanced by tertiary interactions with a s(4)U-G pair. To test the effects of s(2)U and s(4)U substitutions on tertiary interactions, binding to a group I intron ribozyme from mouse-derived Pneumocystis carinii was measured for the hexamers, r(AUGACU), r(AUGACs(2)U), and r(AUGACs(4)U), which mimic the 3' end of the 5' exon. The results suggest that at least one of the carbonyl groups of the 3' terminal U of r(AUGACU) is involved in tertiary interactions with the catalytic core of the ribozyme and/or thio groups change the orientation of a terminal U-G base pair. Thus thio substitutions may affect tertiary interactions. Studies of trans-splicing of 5' exon mimics to a truncated rRNA precursor, however, indicate that thio substitutions have negligible effects on overall reactivity. Therefore, modified bases can enhance the specificity of base pairing while retaining other activities and, thus, increase the specificity of antisense compounds targeting cellular RNA.