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PubMed1981

Alternative conformations of DNA modified by N-2-acetylaminofluorene.

Modification of DNA by the carcinogen N-acetoxy-N-2-acetylaminofluorene gives two adducts, a major one at the C-8 position of guanine and a minor one at the N-2 position with differing conformations. Binding at the C-8 position results in a large distortion of the DNA helix referred to as the "base displacement model" with the carcinogen inserted into the DNA helix and the guanosine displaced to the outside. The result is increased susceptibility to nuclease S1 digestion due to the presence of large, single-stranded regions in the modified DNA. In contrast, the N-2 adduct results in much less distortion of the helix and is less susceptible to nuclease S1 digestion. A third and predominant adduct is formed in vivo, the deacetylated C-8 guanine adduct. The conformation of this adduct has been investigated using the dimer dApdG as a model for DNA. The attachment of aminofluorene (AF) residues introduced smaller changes in the circular dichroism (CD) spectra of dApdG than binding of acetylaminofluorene (AAF) residues. Similarly, binding of AF residues caused lower upfield shifts for the H-2 and H-8 protons of adenine than the AAF residues. These results suggest that AF residues are less stacked with neighboring bases than AAF and induce less distortion in conformation of the modified regions than AAF. An alternative conformation of AAF-modified deoxyguanosine has been suggested based on studies of poly(dG-dC).(dG-dC). Modification of his copolymer with AAF to an extent of 28% showed a CD spectrum that had the characteristics of the left-handed Z conformation seen in unmodified poly(dG-dC).poly(dG-dC) at high ethanol or salt concentrations. Poly(dG-dC).poly(dG-dC) which does not undergo the B to Z transition at high ethanol concentrations, did not show this type of conformational change with high AAF modifications. Differences in conformation were suggested by single-strand specific nuclease S1 digestion and reactivity with anticytidine antibodies. Highly modified poly(GS-dC).poly(DG-dC) was almost completely resistant to nuclease S1 hydrolysis, while, modified DNa and poly(dG).poly(dC) are highly susceptible to digestion. Two possible conformations for deoxyguanosine modified at the C-8 position by AAF are compared depending on whether its position is in alternating purine-pyrimidine sequences or random sequence DNA.

2-Acetylaminofluorene↗

PubMed1989

In situ hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe.

An in situ hybridization technique has been developed for assessing poly(A)+ RNA preservation in routine pathology specimens. The method detects poly-adenylated RNA sequences in tissue sections using a biotinylated polydeoxythymidine (poly d(T)) probe. The probe was prepared from single-stranded 25-30 base oligo d(T) and was biotinylated using the enzyme terminal deoxynucleotide transferase with biotin-11-dUTP and dTTP in the ratio 1:4. The hybridization protocol uses varying concentrations of proteinase K to unmask mRNA sequences and the biotin-labelled hybrids are demonstrated after hybridization under standard conditions by the application of streptavidin and biotinylated alkaline phosphatase. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results have confirmed that the method is specific for poly(A)+ RNA and shows that poly(A)+ RNA can be demonstrated in routine formalin-fixed sections using non-radioactive techniques with retention of morphology. It also provides a means of optimizing the hybridization conditions for specific mRNA probes and produces a staining pattern demonstrating the relative level of poly(A)+ RNA per cell which may reveal new information about cell activity and tissue function.

Base Sequence↗

PubMed2007

Defibrotide for the treatment of hepatic veno-occlusive disease in children.

BACKGROUND: This retrospective report describes experience with defibrotide in children with hepatic veno-occlusive disease (HVOD) following hematopoietic progenitor cell transplant (HPCT) in a single institution. PROCEDURE: Children who had undergone HPCT between February 1999 and June 2001 and between July 2003 and September 2004 and who received defibrotide during their admission were identified. Demographic data and information regarding the clinical course of these patients were abstracted from their health records. RESULTS: Fourteen children (mean age: 9.3 years; range: 0.4-18.1) who underwent HPCT during the study period received defibrotide for the treatment of HVOD; nine were girls. Most patients underwent HPCT for hematologic malignancies (8/14) and received matched unrelated donor transplants (8/14). Conditioning regimens included cyclophosphamide with total body irradiation (5/14) and busulfan followed by cyclophosphamide (7/14). HVOD was diagnosed on transplant day -4 to +33 (median: +10.5); defibrotide was started on transplant day -4 to +40 (median: +12). The median initial defibrotide dose was 33 mg/kg/day (11-40 mg/kg/day); the median maximum defibrotide dose was 38.5 mg/kg/day (11-81 mg/kg/day). The median duration of defibrotide therapy was 16 days (4-37 days). Defibrotide was discontinued due to clinical improvement (9), death (3), drug unavailability (1), and neurological toxicity (1). Gastrointestinal hemorrhage was observed in two patients and intra-cranial hemorrhage was observed in one patient during defibrotide therapy. The survival rate to day +100 was 79%. CONCLUSIONS: Defibrotide appears to be an effective and relatively safe treatment for children with HVOD.

Adolescent↗

PubMed1994

A fission yeast gene encoding a protein that preferentially associates with curved DNA.

We searched for fission yeast (Schizosaccharomyces pombe) proteins that preferentially bind to a synthetic curved DNA sequence, by means of a DNA-binding gel shift assay in the presence of an excess amount of a non-curved DNA sequence as a competitor. We identified such a protein in S. pombe. The protein, thus purified, has an apparent molecular weight of 42,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was suggested that this protein (42 K-protein) recognizes and binds to a curved DNA structure in a given nucleotide sequence, although it also binds to a non-curved DNA sequence with lower affinity. As its putative coding sequence, a 1.9-kilobase genomic DNA from S. pombe was cloned and sequenced. Sequencing of a cDNA clone also revealed the existence of an open reading frame, with no intron, encoding a 381-amino-acid protein with a calculated molecular mass, 41,597. This protein appears to be located in the nucleus. The predicted protein sequence revealed that the 42 K-protein exhibits no significant similarity to any other known proteins, except to a hypothetical protein of Caenorhabditis elegans.

Amino Acid Sequence↗

PubMed1999

Polycations. 5. Inducement of psi-DNA circular dichroism signals for duplex deoxyribonucleotide homopolymers by polycationic strings.

Atypical circular dichroism intensity enhancements previously associated with Psi-DNA condensates are reported for poly(dG-dC):poly(dG-dC) and poly(dA-dT):poly(dA-dT) upon addition to them of a polycationic string salt. Polycationic strings are organic species incorporating several cationic sites in a linear array through the molecule. The effect observed herein with a small molecule model system holds promise for elucidation of the structural details associated with Psi-DNA condensate formation.

Cations↗

PubMed2001

Characterization of parazoanthoxanthin A binding to a series of natural and synthetic host DNA duplexes.

Parazoanthoxanthin A is a fluorescent yellow nitrogenous pigment of the group of zoanthoxanthins, which show a broad range of biological activity. These include, among others, the ability to bind to DNA. In this study we have used a variety of spectroscopic (intrinsic fluorescence emission and UV-spectroscopy) and hydrodynamic techniques (viscometry) to characterize in more detail the binding of parazoanthoxanthin A to a variety of natural and synthetic DNA duplexes in different buffer conditions. Our results reveal the following five significant features: (i) Parazoanthoxanthin A exhibits two modes of DNA binding: One binding mode exhibits properties of intercalation, while the second binding mode is predominantly electrostatic in origin. (ii) The apparent binding "site size" for parazoanthoxanthin A near physiological salt concentration (100 mM NaCl) is in the range of 7 +/- 1 base pairs for natural genomic DNA duplexes (calf thymus and salmon testes DNA) and alternating synthetic polynucleotides (poly[d(AT)]. poly[d(AT)] and poly[d(GC)]. poly[d(GC)]). A slightly larger apparent binding site size of 9 +/- 1 bp was obtained for parazoanthoxanthin A binding to the synthetic homopolymer poly[d(A)]. poly[d(T)]. (iii) Near physiological salt concentration (100 mM NaCl) parazoanthoxanthin A binds with the same approximate binding affinity of 2-5 x 10(5) M(-1) to all DNA polymers studied. (iv) At low salt concentration, parazoanthoxanthin A preferentially binds alternating poly[d(AT)]. poly[d(AT)] and poly[d(GC)]. poly[d(GC)] host duplexes. (v) Parazoanthoxanthin A inhibits DNA polymerase in vitro.

Animals↗

PubMed1997

Thiol-reactive, luminescent Europium chelates: luminescence probes for resonance energy transfer distance measurements in biomolecules.

Lanthanide chelates have recently been shown to be extremely promising luminescence probes for distance measurements in biomolecules using luminescence resonance energy transfer measurements [P. R. Selvin, T. M. Rana, and J. E. Hearst (1994) J. Am. Chem. Soc. 116, 6029-6030; P. R. Selvin, and J. E. Hearst (1994) Proc. Natl. Acad. Sci. USA 91, 10024-10028]. In this work we describe simple procedures for preparing highly fluorescent thiol-reactive europium chelates. These new compounds contain a uv-absorbing coumarin group which sensitizes europium emission, diethylenetriaminepentaacetic acid or triethylenetetraaminehexaacetic acid groups which provide europium chelating function, and a pyridyl disulfide group which allows specific modification of thiol groups. These reagents can be used to label proteins at Cys residues or synthetic oligonucleotides which contain thiol groups. Modification can be reversed easily by treatment with a reducing agent (dithiothreitol). Luminescence energy transfer between these new chelates and CY5 fluorochrome attached to the opposite ends of 15-bp double-stranded DNA was measured to test their usefulness for distance measurements in macromolecules. The distance measured between the chelate (donor) and CY5 (acceptor) was in the range expected for the length of 15-bp DNA. The stability of europium chelates and their conjugates with a protein, the precision of distance measurements using these chelates, possible errors due to intramolecular energy transfer, and the modulation of the R0 value with deuterium oxide were tested. The results obtained fully confirmed the great potential of these new probes for sensitive, simple, and precise distance measurements in biomolecules using luminescence resonance energy transfer.

Base Sequence↗

PubMed2001

Analytical characterization of the conformational transitions of polynucleotides by means of different molecular spectroscopies and multivariate curve resolution.

A general procedure for the study of conformational transitions of polynucleotides is described. The equilibria between different conformations induced by salt, ethidium bromide, and temperature of poly(dG-dC). poly(dG-dC) and induced by salt and temperature of poly(A). poly(U) are investigated using molecular absorption, circular dichroism, and fluorescence spectroscopies. Spectral data obtained from experiments are analyzed by means of a factor analysis method, namely, multivariate curve resolution, which allows possible intermediate states to be detected and the pure spectra and the concentration profiles of all species present in the system to be estimated. This work shows the application of this procedure for the analysis of data matrices obtained in individual experiments but also for the analysis of several data matrices simultaneously.

Circular Dichroism↗

PubMed1993

Recognition of non-B DNA structures by cellular proteins.

By utilising a gel retardation assay, cytoplasmic extracts of Hela cells have been shown to contain a number of polypeptides which bind preferentially to non-B forms of DNA. These polypeptides vary in apparent molecular weights and are present as minor components of the total extract. Similar properties were demonstrated by purified preparations of eukaryotic topoisomerase I and by a DNA binding subunit of the transcription factor NFkB. These results suggest that non-B DNA binding proteins may represent families of proteins which recognise more open forms of DNA as a cognate parameter to their cellular functions.

Chromatography, Affinity↗

PubMed1994

Isolation and identification of aptamers from defibrotide that act as thrombin antagonists in vitro.

This study was designed to isolate and identify aptamer sequences, acting as thrombin antagonists, from defibrotide, a single-stranded DNA fraction. Two different aptamers were identified: 5'-GGTTGGATTGGTTGG-3' and 5'-GGTTGGATCGGTTGG-3'. A third aptamer: 5'-GGATGGATCGGTTGG-3' was found in the PCR product from the double-stranded defibrotide precursor. All aptamers were potent inhibitors of thrombin-induced platelet aggregation, thromboxane biosynthesis, increase in cytosolic Ca++ and fibrin clot formation, effective concentrations being in the nanomolar range. There was no effect on U 46,619 (thromboxane mimetic) or collagen-induced responses. Similar effects were obtained with unfractionated defibrotide while the "nonsense" oligonucleotide 5'-GGTGGTGGTTGTGGT-3' was inactive. The data suggest that antithrombin effects of defibrotide are caused by aptamers.

Animals↗

PubMed1994

Alterations of binding mode and cutting site by G-->I replacement in preferred cleavage sequences 5'-AGG of chromoprotein C-1027.

Replacement of guanosine by inosine at the center position of a target trinucleotide 5'-AGG resulted in greatly diminished DNA cleavage by C-1027 chromophore, indicating that the guanine 2-amino group significantly participates to DNA binding and cleavage by the chromophore of C-1027. This notion was supported by fluorescence titrations of the chromophore with [poly(dG-dC)]2 and [poly(dI-dC)]2. In preferred cleavage sequence 5'-AGG of the enediyne chromophore of C-1027, the G-->I substitution clearly induces change of DNA binding mode and cutting site.

Aminoglycosides↗

PubMed1997

New DNA minor-groove binding molecules with high sequence-selectivities and binding affinities.

New DNA minor-groove binding molecules with high binding affinities and sequence-selectivities are described. The effects of structural changes in ligands with a three-ring backbone on their DNA-binding properties have been studied. Of a pool of eight potential ligands, two showed binding affinities and sequence-selectivities rivaling distamycin. The two best ligands share a common structural motif that involves a para-di-substituted benzene ring flanked by two meta-di-substituted benzene rings.

Animals↗

PubMed1998

The orientation of norfloxacin bound to double-stranded DNA.

Norfloxacin is a widely used antibacterial agent that inhibits DNA gyrase. This fluoroquinolone drug has significant interaction with double-stranded DNA, as judged from absorption and circular dichroism measurements. The mode of binding of norfloxacin to a variety of DNAs and polynucleotides has been investigated by electric linear dichroism. In the presence of calf thymus DNA, the drug chromophore is significantly tilted with respect to the DNA axis. This molecular arrangement contradicts classical intercalation. The orientation of the quinolone drug varies depending on the sequence of the target DNA. Binding to alternating copolymers is largely preferred compared to the corresponding homopolymers. The drug interacts preferentially with poly(dG-dC).(dG-dC) rather than with the other polynucleotides. The deletion of the 2-amino group of guanine (G-->I substitution) or the addition of a methyl group on cytosine residues (C-->methyl-C substitution) affect the drug-DNA interaction. The results show that norfloxacin is capable of interacting with a variety of DNA sequences, possibly via both minor and major groove contacts.

Animals↗

PubMed1998

Polymorphism and disorder of poly(dA).poly(dT) in fibers.

The absence of proportionality between fiber length and interbase axial distance reveals a high degree of disorder in fibers of poly(dA).poly(dT) when the relative humidity (r.h.) varies. According to X-ray patterns, the increase of fiber length does not correspond to any conformational change. The B' structure is present alone and in a better-organized form at higher r.h. We noted a dependence of length values on the direction of variation of the r.h. An unusual minimum of fiber length is observed around 65% r.h. and explained as been due to an amorphous part of the fiber, essentially composed of single strands of poly(dA) and poly(dT). At a temperature higher than 30 degrees C, the transition B'(alpha)-B* takes place but is associated with an important decrease of fiber length in spite of constancy of the helical rise per base pair. It is shown that this behavior is also due to the amorphous part of the fiber and not to a conformational transition.

Models, Chemical↗

PubMed1999

Lipocortin (Annexin) I heterotetramer binds to purine RNA and pyrimidine DNA.

Lipocortin I-like protein with a molecular weight of 94,000 Da as judged by Western analysis was found to bind to ssDNA rather than to dsDNA in a Ca(2+)-dependent manner. This protein was also bound to [(32)P]poly(rA) and [(32)P]poly(rG) as measured by EMSA. Poly(rG), poly(rA), poly(dC), and poly(dT) were competitive against binding of either [(32)P]poly(rA) or [(32)P]poly(rG), while poly(rC), poly(rU), and poly(dA) were less effective binding competitors. The binding of this protein to poly(rA) or poly(rG) was inhibited by immunoprecipitable anti-lipocortin I (calpactin II) and anti-S100 protein antibodies, but not by an anti-Ig antibody. Phospholipids such as phosphatidylserine and phosphatidylinositol enhanced the binding of lipocortin I to poly(rA). Taken together, our present observations suggest that the lipocortin I-S100 protein heterotetramer binds to either purine RNAs or pyrimidine ssDNAs in a Ca(2+)- and phospholipid-dependent manner.

Animals↗

PubMed2000

DNA binding properties of novel distamycin analogs that lack the leading amide unit at the N-terminus.

First examples of distamycin (Dst) analogs which lack hydrogen bond donor or acceptor groups at the N-terminus have been synthesized. The first molecule of this series, which is a bispyrrole peptide, did not exhibit any detectable binding with double-stranded (ds) DNA. However, all other analogs did bind strongly to AT-rich sequences of ds-DNA, with the binding affinities increasing as a function of the number of repeating pyrrole carboxamide units. These results imply that a hydrogen bond donor or acceptor atom per se at the N-terminus is not a prerequisite for DNA binding in the case of pyrrole carboxamide-based Dst analogs. However, in the absence of H-bond donor or acceptor at the N-terminus, a minimum of three pyrrole carboxamide units is necessary for the onset of DNA binding. Beyond this minimum number, the binding affinity increases as a function of the number of pyrrole units, as a result of the greater availability of hydrogen bonding and van der Waals surface. Experiments with poly[d(G-C)] have shown that the presence of the N-terminus formamide group is not inevitable for GC binding of this class of molecules. The observation that the N-terminus formamide unit can be dispensed with suggests that these molecules, which are much easier to synthesize and functionalize, can be used in place of the conventional analogs of distamycin for the development of novel minor groove binders with extended sequence recognition properties.

Binding Sites↗

PubMed2000

DNA annealing and DNA-protein interactions by capillary electrophoresis.

This work deals with annealing of single-stranded DNA and the binding of a serum respond factor to a DNA probe containing specific binding site. Capillary electrophoresis (CE) method is explored and compared with the mobility-shift gel electrophoresis (GE) procedure. The results indicate the CE method offers direct and rapid annealing of the DNA strands. It requires no prior incubation with additives (polynucleotides, proteins) to reduce nonspecific DNA-protein interactions. Unwanted nonspecific interactions are not observed in the CE method. The presence of a fluorescein tag to the DNA probe yields identical results to those with the radioactive label. A fluorescein tag in the CE work can be used without any adverse effects. The dissociation constant (Kd) of this protein-DNA complex by the CE method was similar to those determined by the GE method (approximately 10(-6) M). The proposed method is extremely powerful, highly sensitive, quantitative, and fast. It can determine even very small conformational differences of the DNA probe.

Base Sequence↗

PubMed2001

Unusual features of poly[dT-dG].[dC-dA] stretches in CDS-flanking regions of Trypanosoma cruzi genome.

In trypanosomatids, the mechanisms of gene expression regulation are not yet well understood. The genes are organized into long polycistronic transcription units separated by intergenic regions that may contain the signaling information for nucleic acid processing. Poly-dinucleotides are frequent in these regions and have been proposed to be involved in gene expression regulation. We analyzed their frequency in CDS-flanking sequences of sense strands in Trypanosoma cruzi and established that all but poly[dC-dC], poly[dC-dG], and poly[dG-dG] are significantly more frequent than expected by chance. Poly[dT-dG].[dC-dA] is among the longest and most frequent poly-dinucleotides and shows a remarkable strand asymmetry. Furthermore, electrophoretic mobility shift assays using T. cruzi epimastigotes nuclear extracts demonstrated the existence of at least, one sequence specific single-strand binding activity for each strand. These results strongly suggest that poly[dT-dG].[dC-dA] sequence is involved in regulatory mechanisms of relevance for the parasite biology.

Animals↗