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PubMed1998

Interactions of liposomes and lipid-based carrier systems with blood proteins: Relation to clearance behaviour in vivo.

Liposomes and lipid-based drug delivery systems have been used extensively over the last decade to improve the pharmacological and therapeutic activity of a wide variety of drugs. More recently, this class of carrier systems has been used for the delivery of relatively large DNA and RNA-based drugs, including plasmids, antisense oligonucleotides and ribozymes. Despite recent successes in prolonging the circulation times of liposomes, virtually all lipid compositions studied to date are removed from the plasma compartment within 24h after administration by the cells and tissues of the reticuloendothelial system (RES). Plasma proteins have long been thought to play a critical role in this process but only a few efforts were made to evaluate the relevant importance of plasma protein-liposome interactions in the clearance process. Strategies to increase the bioavailability of liposomal drugs have included altering lipid compositions and charge, increasing lipid doses, and incorporating surface coatings. All of these modifications can influence membrane-protein interactions. In this article, we will focus on our experiences with liposome-blood protein interactions and how alterations in the chemical and physical properties of the carrier system influence the interactions with blood proteins and circulation times.

Journal Article↗
PubMed1983

Nuclear magnetic resonance study on the exchange behavior of the NH-N protons of a ribonucleic acid miniduplex.

The exchange behavior of the guanine N(1) and uracil N(3) protons in the self-complementary hexanucleotide r(ApApGpCpUpU) has been studied at 5 degrees C in 80% H2O/20% D2O by proton NMR. Under these conditions, the hexanucleotide forms a stable miniduplex. The exchange rate of all Watson-Crick NH protons is unaffected by addition of trifluoroethylamine up to 0.07 M. On the other hand, addition of phosphate buffer, pH 6.9, enhances the exchange rate of the uracil N(3) protons of both terminal and internal A X U base pairs but does not influence the exchange rate of the guanine N(1) protons of the central G X C base pairs. Catalysis by increased phosphate concentrations results in an open-limited rate of the internal A X U base pairs with kex = 233 s-1, equivalent to a lifetime of 4.3 ms. The proton exchange of the central G X C is regulated by the opening rate of the central core of the miniduplex. On the other hand, the sensitivity of the exchange rate of internal as well as of terminal A X U base pairs can be explained by their reduced lifetime due to end "fraying" and a subsequent catalysis of the exchange process from the opened state. These results suggest that it may be possible to probe labilized parts of RNAs such as tRNA by gradual addition of the exchange catalyst phosphate and to monitor their exchange rates by proton NMR.

Base Composition↗
PubMed2004

Targeting DNA with novel diphenylcarbazoles.

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.

Base Sequence↗
PubMed1998

Statistical and structural analysis of trp binding sites: comparison of natural and in vitro selected sequences.

Two different modes can be used when the trp repressor binds to trp binding sites. In the "full-site mode" each repressor molecule is bound to a DNA target containing at least two conserved five base pair tracts separated by eight base pairs. The binding of the repressor to natural trp operators is of this kind. In the "half-site mode" two repressor molecules are sequence-specifically bound, with infinite cooperativity, to two abutting DNA pentamers. We present evidence suggesting that the sequences obtained by a recent in vitro selection assay (Czernik et al. J. Biol. Chem. 269, 27869-27875, 1994) were selected by the binding of two repressor molecules, and that the repressor is bound to most of these sequences using the half-site mode. Using the results of the selection assay, and the set of natural trp binding sites, we characterize the different sequence requirements of the "full-site" versus the "half-site" binding modes. A statistical analysis of the information content of these binding sites shows that functional information on protein binding modes can be extracted from a set of DNA binding sites by comparing the information content of two different DNA populations, or sub-populations. Furthermore, it shows that the binding of proteins to sequences selected by a functional in vitro assay do not necessarily mimic the binding of the protein to the natural targets, even if the information content is similar in the two DNA target populations, i.e., even if the stringency of the selection assay is adequate for locating natural-like sequences. In addition, we show that the structural requirements for protein-DNA interactions can be achieved by different conformations at the base-pair level. Differences in the structural characteristics of different base-pair steps can be used to determine the binding mode and differential binding affinity, which can be utilized in the regulation of several binding sites by a single specific protein.

Apoproteins↗
PubMed1992

Molecular and immunological characterization of ADP-ribosylarginine hydrolases.

Mono-ADP-ribosylation is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the forward and reverse reactions, respectively. Hydrolase activities were present in a variety of animal species, with the highest specific activities found in rat and mouse brain, spleen, and testis. Rat and mouse hydrolases were dithiothreitol- and Mg(2+)-dependent, whereas the bovine and guinea pig enzymes were dithiothreitol-independent. A rat brain hydrolase was purified approximately 20,000-fold and represented the major approximately 39-kDa protein on denaturing gels. Immunoaffinity-purified rabbit polyclonal antibodies reacted with 39-kDa proteins from turkey erythrocytes and rat, mouse, and calf brains. A rat brain cDNA library was screened using oligonucleotide and polymerase chain reaction-generated cDNA probes. Inserts from two overlapping clones yielded a composite sequence that included a 1086-base pair open reading frame, which contained amino acid sequences found in the purified hydrolase. A hydrolase fusion protein, synthesized in Escherichia coli, reacted with anti-39-kDa polyclonal antibodies and exhibited Mg(2+)- and dithiothreitol-dependent hydrolase activity. A coding region cDNA hybridized readily to a 1.7-kilobase band in rat and mouse poly(A)+ RNA, but poorly to bovine, chicken, rabbit, and human poly(A)+ RNA. The immunological and molecular biological data are consistent with partial conservation of hydrolase structure across animal species.

Amino Acid Sequence↗
PubMed2000

Microbial ecology of nitrifying bacteria in wastewater treatment process examined by fluorescence in situ hybridization.

The microbial ecology of nitrifying bacteria in various types of wastewater treatment processes and the dynamic response of the microbial ecology in biofilms were investigated using fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. Nitrifying bacteria were found to exhibit various organizational forms under different conditions of substrate composition and concentration. Ammonia-oxidizing bacteria were dominant in ammonia-rich inorganic wastewater, while heterotrophic bacteria and ammonia-oxidizing bacteria were localized at different positions in the biofilm in organic wastewater. The dynamics of the microbial ecology in the biofilm with regard to the spatial distribution of ammonia-oxidizing bacteria and heterotrophic bacteria caused by a gradual change in substrate composition was successfully monitored by FISH analysis.

Journal Article↗
PubMed1990

The ANB1 locus of Saccharomyces cerevisiae encodes the protein synthesis initiation factor eIF-4D.

The Saccharomyces cerevisiae anaerobic gene (ANB1) is negatively regulated both by oxygen and heme. We have shown recently that an upstream repressor site located in the 5'-flanking region of this gene controls its expression (Mehta, K.D., and Smith, M. (1989) J. Biol. Chem. 265, 8670-8675). In this paper, we present the complete genomic sequence of the ANB1 locus of S. cerevisiae. The ANB1 locus encodes a protein of 157 residues with an Mr of 17, 134. The deduced amino acid sequence of the ANB1 gene product shows strikingly extensive sequence and structural homology (63.5% identical residues and an additional 15% conservative substitutions) to the 154-amino-acid-long human and rabbit eukaryotic translation initiator factor (eIF)-4D. Factor eIF-4D is the only known mammalian protein that undergoes a unique post-translational modification of Lys-50 to the amino acid hypusine, and interestingly the same lysine is also present in the ANB1 gene product. Results presented provide strong evidence that the ANB1 locus that encodes a transcript, tr-2, and a second locus encoding a transcript, tr-1, together encode two forms of yeast eIF-4D. Interestingly, heme regulates both the loci in an opposite manner; as a result it can dictate the isoform available under conditions of high and low oxygen tension. The ROX1 locus of S. cerevisiae is known to regulate CYC1, COXVb, and ANB1 genes at the transcriptional level; the ROX1 locus thus regulates all known anaerobically expressed genes that are involved in different cellular functions such as respiration and protein synthesis.

Amino Acid Sequence↗
PubMedDate not supplied

[Possible conformations of nucleic acid fragments containing the base pair guanine:uracil].

By using classical potential functions, nonbound interaction energy calculations are carried out on the deoxydinucleoside phosphate complexes dUpdA:dUpdG and dUpdU:dGpdA. All dihedral and bond angles, except those of the nitrogen bases, are varied. On the basis of the calculations possible conformations of nucleic acid fragments containing G:U base pair are proposed. Dihedral angles of the conformations are close to those of regular Watson-Crick helices while interaction energies are 3-4 kcal/mole higher than that of A:U containing fragment conformations.

Base Composition↗
PubMed1997

Affinity capillary electrophoresis using DNA conjugates.

An oligonucleotide ((dT)12)-immobilized capillary was prepared for the affinity capillary electrophoresis of DNA. Oligonucleotides ((dA)6 and (dT)6) which had the same chain length but had different base composition were separated in high resolution in the presence of Mg2+ ion. Whereas (dA)6 eluted slightly earlier than (dT)6 in a conventional polyacrylamide-coated capillary, the order of their mobilities was reversed in the (dT)12-modified system, suggesting the complementary hydrogen bondings between (dA)6 and the immobilized (dT)12.

Base Composition↗
PubMed1998

Oligonucleotide bias in Bacillus subtilis: general trends and taxonomic comparisons.

We present a general analysis of oligonucleotide usage in the complete genome of Bacillus subtilis . Several datasets were built in order to assign various biological contexts to the biased use of words and to reveal local asymmetries in word usage that may be coupled with replication, the control of gene expression and the restriction/modification system. This analysis was complemented by cross-comparisons with the complete genomes of Escherichia coli , Haemophilus influenzae and Methanococcus jannaschii . We have observed a large number of biased oligonucleotides for words of size up to 8, throughout the datasets and species, indicating that such long strict words play an important role as biological signals. We speculate that some of them are involved in interactions with DNA and/or RNA polymerases. An extensive analysis of palindrome abundances and distributions provides the surprising result that prophage-like elements embedded in the genome exhibit a smaller avoidance of restriction sites. This may reinforce a recently proposed hypothesis of a selfish gene phenomena in the transfer of restriction/modification systems in bacteria.

Bacillus subtilis↗
PubMed1998

Inhibition of ribosomal subunit association and protein synthesis by oligonucleotides corresponding to defined regions of 18S rRNA and 5S rRNA.

Strong complementarity between a conserved sequence near the 3' end of 18S (16S) rRNA of the small ribosomal subunit and a conserved sequence in the 5S rRNA of the large ribosomal subunit supported the suggestion that base-paired interaction between the two RNA molecules could be responsible for the reversible association of ribosomal subunits during protein synthesis. If this were true then oligonucleotides corresponding to defined regions of the 18S and 5S rRNAs should have profound effects on the association of ribosomal subunits and protein synthesis. In this report we show that oligonucleotides, corresponding to a defined region of eukaryotic 18S rRNA, when bound to wheat embryo 60S ribosomal subunits, inhibited association with 40S ribosomal subunits and also inhibited in vitro protein synthesis. Similarly oligonucleotides corresponding to a defined region of 5S rRNA when bound to 40S ribosomal subunits also inhibited the formation of 80S ribosomes and in vitro protein synthesis. The minimum sequences responsible for the inhibition of ribosomal subunit association and in vitro protein synthesis corresponded to the 5' strand of the m2(6)A m2(6)A hairpin structure near the 3' end of 18S rRNA and nucleotides 91-100 of 5S rRNA which are complementary to each other. Sequences at identical positions of Escherichia coli 16S and 5S rRNAs are also complementary to each other.

Base Composition↗
PubMed2000

[Cleavage of RNA in hybrid duplexes by E. coli ribonuclease H. II. Substrate properties of nucleotides containing non-nucleotide linkers].

The 20-mer bridged oligodeoxynucleotides containing short oligomers joined by the hexamethylenediol and hexaethylene glycol linkers were shown to form complementary DNA/DNA and RNA/DNA complexes whose thermostability depends on the length and number of the nonnucleotide linkers. Hybrid complexes of the bridged oligonucleotides proved to be substrates for the E. coli ribonuclease H. The presence of one-three nonnucleotide linkers in a 20-mer decreased the hydrolysis efficacy only 1.2-1.4-fold. It is the composition of the RNA cleavage products that was influenced the most significantly by the nonnucleotide linkers. RNase H simultaneously hydrolyzed the RNA 3'-ends of each hybrid duplex involving a bridged oligonucleotide. The presence of an inverted 3'-3'-phosphodiester bond at the 3'-end of the oligodeoxyribonucleotide only slightly affected the RNase H activity.

DNA↗
PubMed2004

Application and validation of DNA microarrays for the 16S rRNA-based analysis of marine bacterioplankton.

An oligonucleotide probe-based DNA microarray was evaluated for its ability to detect 16S rRNA targets in marine bacterioplankton samples without prior amplification by polymerase chain reaction (PCR). The results obtained were compared with those of quantitative fluorescence in situ hybridization (FISH). For extraction and direct labelling of total RNA, a fast and efficient protocol based on commercially available kits was established. A set of redundant and hierarchically structured probes was applied, and specificity of hybridization was assessed by additional control oligonucleotides comprising single central mismatches. The protocol was initially tested by microarray analysis of bacterial pure cultures. Complete discrimination of all control oligonucleotides was achieved, indicating a high degree of hybridization specificity. In a co-culture, abundant members were detected by microarray analysis, but signal ratios of positive probes did not correlate well with quantitative data from FISH experiments. A marine picoplankton sample from the German Bight was analysed. Bacterial populations with relative abundances of at least 5% were detected by hybridizing 0.1 microg of total RNA extracted from a sample of 375 ml equivalent to 4.1 x 10(8) cells. Our results demonstrate that major populations of marine bacterioplankton can be identified by microarray analysis in a fast and reliable way, even in relatively low volumes of sea water.

Bacteria↗
PubMed1990

Local variability and base sequence effects in DNA crystal structures.

The importance and usefulness of local doublet parameters in understanding sequence dependent effects has been described for A- and B-DNA oligonucleotide crystal structures. Each of the two sets of local parameters described by us in the NUPARM algorithm, namely the local doublet parameters, calculated with reference to the mean z-axis, and the local helical parameters, calculated with reference to the local helix axis, is sufficient to describe the oligonucleotide structures, with the local helical parameters giving a slightly magnified picture of the variations in the structures. The values of local doublet parameters calculated by NUPARM algorithm are similar to those calculated by NEWHELIX90 program, only if the oligonucleotide fragment is not too distorted. The mean values obtained using all the available data for B-DNA crystals are not significantly different from those obtained when a limited data set is used, consisting only of structures with a data resolution of better than 2.4 A and without any bound drug molecule. Thus the variation observed in the oligonucleotide crystals appears to be independent of the quality of their crystallinity. No strong correlation is seen between any pair of local doublet parameters but the local helical parameters are interrelated by geometric relationships. An interesting feature that emerges from this analysis is that the local rise along the z-axis is highly correlated with the difference in the buckle values of the two basepairs in the doublet, as suggested earlier for the dodecamer structures (Bansal and Bhattacharyya, in Structure & Methods: DNA & RNA, Vol. 3 (Eds., R.H. Sarma and M.H. Sarma), pp. 139-153 (1990)). In fact the local rise values become almost constant for both A- and B-forms, if a correction is applied for the buckling of the basepairs. In B-DNA the AA, AT, TA and GA basepair sequences generally have a smaller local rise (3.25 A) compared to the other sequences (3.4 A) and this seems to be an intrinsic feature of basepair stacking interaction and not related to any other local doublet parameter. The roll angles in B-DNA oligonucleotides have small values (less than +/- 8 degrees), while mean local twist varies from 24 degrees to 45 degrees. The CA/TG doublet sequences show two types of preferred geometries, one with positive roll, small positive slide and reduced twist and another with negative roll, large positive slide and increased twist.(ABSTRACT TRUNCATED AT 400 WORDS)

Algorithms↗
PubMed2005

Ecological differentiation within a cosmopolitan group of planktonic freshwater bacteria (SOL cluster, Saprospiraceae, Bacteroidetes).

Members of the monophyletic SOL cluster are large filamentous bacteria inhabiting the pelagic zone of many freshwater habitats. The abundances of SOL bacteria and compositions of SOL communities in samples from 115 freshwater ecosystems around the globe were determined by fluorescence in situ hybridization with cluster- and subcluster-specific oligonucleotide probes. The vast majority (73%) of sampled ecosystems harbored SOL bacteria, and all three previously described SOL subclusters (LD2, HAL, and GKS2-217) were detected. The morphometric and chemicophysical parameters and trophic statuses of ecosystems were related to the occurrence and subcluster-specific composition of SOL bacteria by multivariate statistical methods. SOL bacteria did not occur in acidic lakes (pH < 6), and their abundance was negatively related to high trophy and pH. The subcluster-specific variation in the compositions of SOL communities could be related to the pH, electrical conductivity, altitude, and trophic status of ecosystems. All three known SOL subclusters differed in respect to their tolerated ranges of pH and conductivity. Complete niche separation was observed between the vicarious subclusters GKS2-217 and LD2; the former occurred in soft-water lakes, whereas the latter was found in a broad range of hard-water habitats. The third subgroup (HAL) showed a wide environmental tolerance and was usually found sympatrically with the LD2 or GKS2-217 subcluster. Ecological differentiation of SOL bacteria at the subcluster level was most probably driven by differential adaptation to water chemistry. The distribution of the two vicarious taxa seems to be predominantly controlled by the geological backgrounds of the catchment areas of the habitats.

Animals↗
PubMed1993

Both O4-methylthymine and O4-ethylthymine preferentially form alkyl T.G pairs that do not block in vitro replication in a defined sequence.

The mutagenic potential of O4-methylthymine (m4T) and O4-ethylthymine (e4T) was determined by a primer extension assay on a 25mer oligonucleotide containing a single site-specifically incorporated modified thymine. The e4T-containing oligonucleotide was prepared by using a new synthetic procedure suitable for large alkyl groups on thymine. The second-order rate constants, K(app)m and V(rel)max, permitted calculation of the frequency of formation and extension of modified base pairs compared to Watson-Crick pairing. With both m4T and e4T, the T.G type pairing was formed at least 10-fold more frequently than the nonmutagenic alkyl T.A pairing. However, there was a small but reproducible preference for m4T.G pairing. In both cases T-->C transitions would result. There was no evidence for formation of alkyl T.C or T.T. These data suggest that reported T-->A transversions by ethylation are not likely to result from O4-alkylthymine. In contrast to insertion, extension beyond alkylthymine under kinetic conditions did not occur with alkyl T.A. but only with the alkyl T.G termini. For this latter T.G type pairing, the larger ethyl group did not hinder extension compared to that of the methyl group, in the sequence studied. Under non-limiting conditions of dNTP concentration and time, complete replication could be demonstrated for both methyl- and ethyl-containing oligonucleotides. We conclude that the increase in size of the alkyl group from methyl to ethyl does not significantly affect the mutagenic potential and type of mutations of O4-alkylthymine in vitro.

Adenosine Triphosphate↗
PubMed2000

Phylogenetic analysis of and oligonucleotide probe development for eikelboom type 021N filamentous bacteria isolated from bulking activated sludge.

Fifteen filamentous strains, morphologically classified as Eikelboom type 021N bacteria, were isolated from bulking activated sludges. Based on comparative 16S ribosomal DNA (rDNA) sequence analysis, all strains form a monophyletic cluster together with all recognized Thiothrix species (88.3 to 98.7% 16S rDNA sequence similarity) within the gamma-subclass of Proteobacteria. The investigated Eikelboom type 021N isolates were subdivided into three distinct groups (I to III) demonstrating a previously unrecognized genetic diversity hidden behind the uniform morphology of the filaments. For in situ detection of these bacteria, 16S rRNA-targeted oligonucleotide probes specific for the entire Eikelboom type 021N-Thiothrix cluster and the Eikelboom type 021N groups I, II, and III, respectively, were designed, evaluated, and successfully applied in activated sludge.

Base Composition↗
PubMed1998

Structural and kinetic characterization of an acyl transferase ribozyme.

We have previously isolated, by in vitro selection, an acyl-transferase ribozyme that is capable of transferring a biotinylated methionyl group from the 3' end of a hexanucleotide substrate to its own 5'-hydroxyl. Comparison of the sequences of a family of evolved derivatives of this ribozyme allowed us to generate a model of the secondary structure of the ribozyme. The predicted secondary structure was extensively tested and confirmed by single-mutant and compensatory double-mutant analyses. The role of the template domain in aligning the acyl-donor oligonucleotide and acyl-acceptor region of the ribozyme was confirmed in a similar manner. The significance of different domains of the ribozyme structure and the importance of two tandem G:U wobble base pairs in the template domain were studied by kinetic characterization of mutant ribozymes. The wobble base pairs contribute to the catalytic rate enhancement, but only in the context of the complete ribozyme; the ribozyme in turn alters the metal binding properties of this site. Competitive inhibition experiments with unacylated substrate oligonucleotide are consistent with the ribozyme acting to stabilize substrate binding to the template, while negative interactions with the aminoacyl portion of the substrate destabilize binding.

Acyltransferases↗