-
Nucleic Acids Research Nov 2010Herein, we study the nanomechanical characteristics of single DNA molecules in the presence of DNA binders, including intercalating agents (ethidium bromide and...
Herein, we study the nanomechanical characteristics of single DNA molecules in the presence of DNA binders, including intercalating agents (ethidium bromide and doxorubicin), a minor groove binder (netropsin) and a typical alkylating damaging agent (cisplatin). We have used magnetic tweezers manipulation techniques, which allow us to measure the contour and persistence lengths together with the bending and torsional properties of DNA. For each drug, the specific variations of the nanomechanical properties induced in the DNA have been compared. We observed that the presence of drugs causes a specific variation in the DNA extension, a shift in the natural twist and a modification of bending dependence on the imposed twist. By introducing a naive model, we have justified an anomalous correlation of torsion data observed in the presence of intercalators. Finally, a data analysis criterion for discriminating between different molecular interactions among DNA and drugs has been suggested.
Topics: Antineoplastic Agents, Alkylating; Biomechanical Phenomena; Cisplatin; DNA; Doxorubicin; Ethidium; Intercalating Agents; Ligands; Magnetics; Netropsin; Nucleic Acid Conformation
PubMed: 20601682
DOI: 10.1093/nar/gkq597 -
ACS Omega Oct 2022The experiments described here examined the effects of reaction conditions, various additives, and local sequence on the formation and stability interstrand cross-links...
Effects of Local Sequence, Reaction Conditions, and Various Additives on the Formation and Stability of Interstrand Cross-Links Derived from the Reaction of an Abasic Site with an Adenine Residue in Duplex DNA.
The experiments described here examined the effects of reaction conditions, various additives, and local sequence on the formation and stability interstrand cross-links (ICLs) derived from the reaction of an apurinic/apyrimidinic (AP) site with the exocyclic amino group of an adenine residue on the opposing strand in duplex DNA. Cross-link formation was observed in a range of different buffers, with faster formation rates observed at pH 5. Inclusion of the base excision repair enzyme alkyladenine DNA glycosylase (hAAG) which binds tightly to AP-containing duplexes decreased, but did not completely prevent, formation of the dA-AP ICL. Formation of the dA-AP ICL was not altered by the presence of the biological metal ion Mg or the biological thiol, glutathione. Several organocatalysts of imine formation did not enhance the rate of dA-AP ICL formation. Duplex length did not have a large effect on dA-AP yield, so long as the melting temperature of the duplex was not significantly below the reaction temperature (the duplex must remain hybridized for efficient ICL formation). Formation of the dA-AP ICL was examined in over 40 different sequences that varied the neighboring and opposing bases at the cross-linking site. The results indicate that ICL formation can occur in a wide variety of sequence contexts under physiological conditions. Formation of the dA-AP ICL was strongly inhibited by the aldehyde-trapping agents methoxyamine and hydralazine, by NaBHCN, by the intercalator ethidium bromide, and by the minor groove-binding agent netropsin. ICL formation was inhibited to some extent in bicarbonate and Tris buffers. The dA-AP ICL showed substantial inherent stability under a variety of conditions and was not a substrate for AP-processing enzymes APE1 or Endo IV. Finally, we characterized cross-link formation in a small (11 bp) stem-loop (hairpin) structure and in DNA-RNA hybrid duplexes.
PubMed: 36278095
DOI: 10.1021/acsomega.2c05736 -
Journal of Nucleic Acids May 2010Guanine-rich nucleic acid sequences can adopt G-quadruplex structures stabilized by layers of four Hoogsteen-paired guanine residues. Quadruplex-prone sequences are...
Guanine-rich nucleic acid sequences can adopt G-quadruplex structures stabilized by layers of four Hoogsteen-paired guanine residues. Quadruplex-prone sequences are found in many regions of human genome and in the telomeres of all eukaryotic organisms. Since small molecules that target G-quadruplexes have been found to be effective telomerase inhibitors, the identification of new specific ligands for G-quadruplexes is emerging as a promising approach to develop new anticancer drugs. Distamycin A is known to bind to AT-rich sequences of duplex DNA, but it has recently been shown to interact also with G-quadruplexes. Here, isothermal titration calorimetry (ITC) and NMR techniques have been employed to characterize the interaction between a dicationic derivative of distamycin A (compound 1) and the [d(TGGGGT)](4) quadruplex. Additionally, to compare the binding behaviour of netropsin and compound 1 to the same target, a calometric study of the interaction between netropsin and [d(TGGGGT)](4) has been performed. Experiments show that netropsin and compound 1 are able to bind to [d(TGGGGT)](4) with good affinity and comparable thermodynamic profiles. In both cases the interactions are entropically driven processes with a small favourable enthalpic contribution. Interestingly, the structural modifications of compound 1 decrease the affinity of the ligand toward the duplex, enhancing the selectivity.
PubMed: 20725616
DOI: 10.4061/2010/247137 -
FEBS Letters Oct 1994The influence of netropsin (Nt), distamycin A (Dst-3) and chromomycin A3 (CHR) on the binding of gyrase from Streptomyces noursei to an 162 bp-fragment of pBR 322...
The influence of netropsin (Nt), distamycin A (Dst-3) and chromomycin A3 (CHR) on the binding of gyrase from Streptomyces noursei to an 162 bp-fragment of pBR 322 containing a strong gyrase cleavage site and on the gyrase mediated cleavage of this fragment was analyzed. Binding of the enzyme to the fragment is effectively inhibited by the GC-specific drug CHR, but poorly influenced by Dst-3, while Nt is ineffective. Cleavage of the fragment catalysed by the enzyme is inhibited by all three ligands but to different extent. Dst-3 and Nt inhibit the enzyme cleavage reaction at 20- or 250-fold higher concentration than that required for CHR. The inhibitory mechanism of CHR on gyrase-DNA binding and cleavage may be related to a competitive interaction of the ligand to GC sequences located at and around the gyrase cleavage site. The fact that AT-specific minor groove binders Dst-3 and Nt poorly inhibit the binding of gyrase to the fragment due to the low amount of the AT basepair sequences contained in the fragment and their inhibitory influence on the cleavage step underlines the role of the DNA minor groove during enzyme action.
Topics: Base Sequence; Chromomycin A3; DNA Topoisomerases, Type II; DNA, Bacterial; DNA-Binding Proteins; Distamycins; Hydrolysis; Molecular Sequence Data; Netropsin; Protein Binding; Streptomyces
PubMed: 7926028
DOI: 10.1016/0014-5793(94)00998-8 -
European Journal of Biochemistry Jul 1971
Topics: Absorption; Animals; Anti-Bacterial Agents; Binding Sites; Carcinoma, Ehrlich Tumor; Cattle; DNA; Escherichia coli; Hot Temperature; In Vitro Techniques; Peptides; Phosphates; Pyrroles; RNA; RNA Nucleotidyltransferases; Spectrum Analysis; Streptomyces; Temperature; Ultraviolet Rays
PubMed: 4935203
DOI: 10.1111/j.1432-1033.1971.tb01466.x -
Nucleic Acids Research Apr 2015DNA-binding and RNA-binding proteins are usually considered 'undruggable' partly due to the lack of an efficient method to identify inhibitors from existing small...
DNA-binding and RNA-binding proteins are usually considered 'undruggable' partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein-nucleic acids interactions based on protein-DNA or protein-RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobility-group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2-DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2-DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2-DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.
Topics: 3T3-L1 Cells; Adipogenesis; Animals; DNA; DNA-Binding Proteins; Enzyme-Linked Immunosorbent Assay; HMGA2 Protein; High-Throughput Screening Assays; Mice; Netropsin; RNA-Binding Proteins
PubMed: 25653160
DOI: 10.1093/nar/gkv069 -
Genetics Jan 1978Mutants have been isolated in S. cerevisiae with the phenotype of growth on pyruvate but not on glucose, or growth on rich medium with pyruvate but inhibition by...
Mutants have been isolated in S. cerevisiae with the phenotype of growth on pyruvate but not on glucose, or growth on rich medium with pyruvate but inhibition by glucose. Screening of mutagenized cultures was either without an enrichment step, or after enrichment using the antibiotic netropsin (Young et al. 1976) or inositol starvation (Henry, Donahue and Culbertson 1975). One class of mutants lacked pyruvate kinase (pyk), another class had all the enzymes of glycolysis, and one mutant lacked phosphoglucose isomerase (pgi, Maitra 1971). Partial reversion of pyruvate kinase mutants on rich medium containing glucose gave double mutants now also lacking hexokinase (hxk), phosphofructokinase (fk), or several enzymes of glycolysis (gcr). In diploids the mutations were recessive. pyk, pgi, pfk, and gcr segregated 2:2 from their wild-type alleles. PYK hxk, PYK pfk, and PYK gcr segregrants grew on glucose.
Topics: Glucose-6-Phosphate Isomerase; Glycolysis; Hexokinase; Mutation; Phosphofructokinase-1; Pyruvate Kinase; Saccharomyces cerevisiae
PubMed: 147195
DOI: 10.1093/genetics/88.1.1 -
Journal of the American Chemical Society Nov 2012A variety of solution methods exist for analysis of interactions between small molecule ligands and nucleic acids; however, accomplishing this task economically at the...
A variety of solution methods exist for analysis of interactions between small molecule ligands and nucleic acids; however, accomplishing this task economically at the scale of hundreds to thousands of sequences remains challenging. Surface assays offer a prospective solution through array-based multiplexing, capable of mapping out the full sequence context of a DNA/ligand interaction in a single experiment. However, relative to solution assays, accurate quantification of DNA/ligand interactions in a surface format must contend with limited understanding of molecular activities and interactions at a solid-liquid interface. We report a surface adaptation of a solution method in which shifts in duplex stability, induced by ligand binding and quantified from melting transitions, are used for thermodynamic analysis of DNA/ligand interactions. The results are benchmarked against solution calorimetric data. Equilibrium operation is confirmed through superposition of denaturation/hybridization transitions triggered by heating and cooling. The antibiotic compound netropsin, which undergoes electrostatic and sequence-specific minor groove interactions with DNA, is used as a prototypical small molecule. DNA/netropsin interactions are investigated as a function of ionic strength and drug concentration through electrochemical tracing of surface melt transitions. Comparison with solution values finds excellent agreement in free energy, though reliable separation into enthalpic and entropic contributions proves more difficult. The results establish key guidelines for analysis of DNA-ligand interactions via reversible melting denaturation at surfaces.
Topics: Adsorption; Anti-Bacterial Agents; DNA; Ligands; Netropsin; Osmolar Concentration; Surface Properties; Thermodynamics
PubMed: 23046441
DOI: 10.1021/ja3066368 -
Acta Poloniae Pharmaceutica 2008Inhibitory effects of nine carbocyclic DNA minor groove binders on amidolytic activities of plasmin, trypsin and urokinase were examined. Some of the studied compounds...
Inhibitory effects of nine carbocyclic DNA minor groove binders on amidolytic activities of plasmin, trypsin and urokinase were examined. Some of the studied compounds affected plasmin or trypsin activity, but not urokinase activity. One of the pentamidine analogues (5) and two bis-netropsin like compounds (6, 8) were potent inhibitors of plasmin (IC50 equals 90 and 100 microM), whereas an analogue of netropsin (2) was trypsin inhibitor (IC50 = 100 microM).
Topics: Amines; Fibrinolysin; Netropsin; Pentamidine; Structure-Activity Relationship; Trypsin Inhibitors; Urokinase-Type Plasminogen Activator
PubMed: 18666427
DOI: No ID Found -
The Biochemical Journal Apr 1987
Review
Topics: Base Sequence; DNA; Diminazene; Echinomycin; Intercalating Agents; Macromolecular Substances; Models, Molecular; Netropsin; Nogalamycin; Pharmaceutical Preparations
PubMed: 3038075
DOI: 10.1042/bj2430001