Sequence-specific binding of [N-MeCys3,N-MeCys7]TANDEM to TpA.
ABSTRACT: The sequence selective binding of [N-MeCys3,N-MeCys7]TANDEM to DNA has been studied by footprinting experiments on DNA fragments containing the self-complementary sequences CGCGATATCGCG, CGCGTATACGCG, CGCGTTAACGCG and CGCGAATTCGCG. DNAase I and micrococcal nuclease reveal drug-induced footprints with the central sequences ATAT, TATA and TTAA, but not AATT, suggesting that the ligand binds to the dinucleotide TpA. The ligand renders certain adenines hyper-reactive to diethyl pyrocarbonate. These are observed with ATAT, TATA and TTAA, but not AATT, and are located both within, and distal to, the TpA-binding sites.
Project description:We have examined the dissociation of [N-MeCys3,N-MeCys7]TANDEM, an AT-selective bifunctional intercalator, from TpA sites in mixed-sequence DNAs by a modification of the footprinting technique. Dissociation of complexes between the ligand and radiolabelled DNA fragments was initiated by adding a vast excess of unlabelled calf thymus DNA. Portions of this mixture were subjected to DNAse I footprinting at various times after adding the competitor DNA. Dissociation of the ligand from each site was seen by the time-dependent disappearance of the footprinting pattern. Within a natural DNA fragment (tyrT) the ligand dissociates from TTAT faster than from ATAT. We found that the stability of complexes with isolated TpA steps decreases in the order ATAT > TTAA > TATA. Dissociation from each of these sites is much faster than from longer regions of (AT)n. These results confirm the requirement for A and T base-pairs surrounding the TpA step and suggest that the interaction is strongest with regions of alternating AT, possibly as a result of its unusual structure. The ligand dissociates more slowly from the centre of (AT)n tracts than from the edges, suggesting that variations in dissociation rate arise from sequence-dependent variations in local DNA structure.
Project description:With the increasing number and variations of genome sequences available, control of gene expression with synthetic, cell-permeable molecules is within reach. The variety of sequence-specific binding agents is, however, still quite limited. Many minor groove binding agents selectivity recognize AT over GC sequences but have less ability to distinguish among different AT sequences. The goal with this article is to develop compounds that can bind selectively to different AT sequences. A number of studies indicate that AATT and TTAA sequences have significantly different physical and interaction properties and different requirements for minor groove recognition. Although it has been difficult to get minor groove binding at TTAA, DB293, a phenyl-furan-benzimidazole diamidine, was found to bind as a strong, cooperative dimer at TTAA but with no selectivity over AATT. In order to improve selectivity, we made modifications to each unit of DB293. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1003, a furan-furan-benzimidazole diamidine, binds strongly to TTAA as a dimer and has selectivity (K(TTAA)/K(AATT)=6). CD and DNase I footprinting studies confirmed the preference of this compound for TTAA. In summary, (i) a favorable stacking surface provided by the pi system, (ii) H-bond donors to interact with TA base pairs at the floor of the groove provided by a benzimidazole (or indole) -NH and amidines, and (iii) appropriate curvature of the dimer complex to match the curvature of the minor groove play important roles in differentiating the TTAA and AATT minor grooves.
Project description:The interaction of echinomycin with a kinetoplast DNA fragment which contains phased runs of adenine residues has been examined by various footprinting techniques. DNAase I footprinting confirms that all drug-binding sites contain the dinucleotide CpG. However, not all such sequences are protected. Three sites, each of which is located between two adenine tracks in the sequence GCGA, are not protected from DNAase I attack. Enhanced cleavage by DNAase I, DNAase II and micrococcal nuclease is observed in regions surrounding drug-binding sites. The results suggest that echinomycin alters the conformation of the AT tracks, making them more like an average DNA structure. Echinomycin renders adenine residues in the sequence CGA hyper-reactive to diethyl pyrocarbonate.
Project description:An analogue of the DNA-binding compound Hoechst 33258, in which the piperazine ring has been replaced by an imidazoline group, has been cocrystallized with the dodecanucleotide sequence d(CGCGAATTCGCG)2. The structure has been solved by X-ray diffraction analysis and has been refined to an R-factor of 19.7% at a resolution of 2.0 A. The ligand is found to bind in the minor groove, at the central four AATT base pairs of the B-DNA double helix, with the involvement of a number of van der Waals contacts and hydrogen bonds. There are significant differences in minor groove width for the two compounds, along much of the AATT region. In particular this structure shows a narrower groove at the 3' end of the binding site consistent with the narrower cross-section of the imidazole group compared with the piperazine ring of Hoechst 33258 and therefore a smaller perturbation in groove width. The higher binding affinity to DNA shown by this analogue compared with Hoechst 33258 itself, has been rationalised in terms of these differences.
Project description:The pursuit of small molecules that bind to DNA has led to the discovery of selective and potent antitrypanosomal agents, specifically 4,4'-bis(imidazolinylamino)- and 4,4'-bis(guanidino)diphenylamine compounds, CD27 and CD25, respectively. Although the antitrypanosomal properties of these compounds have been characterized, further development of this series of compounds requires assessment of their DNA site selectivities and affinities. Toward this end, both compounds have been analyzed and found to selectively bind AT sequences. However, CD27 was found to bind with higher affinity to 5'-AATT than 5'-ATAT while CD25 bound more weakly but equally well to either sequence. To detail the nature of its interactions with DNA, the crystal structure of CD27, bound to its preferred DNA-binding site 5'-AATT within a self-complementary oligonucleotide, 5'-d(CTTAATTCGAATTAAG), was determined at 1.75 A using a host-guest approach. Although CD27 is predicted to be highly twisted in its energy-minimized state, it adopts a more planar crescent shape when bound in the minor groove of the DNA. Interactions of CD27 with 5'-AATT include bifurcated hydrogen bonds, providing a basis for selectivity of this site, and favorable van der Waals interactions in a slightly widened minor groove. Thus, an induced fit results from conformational changes in both the ligand and the DNA. Our studies suggest a basis for understanding the mechanism of the antitrypanosomal activity of these symmetric diphenylamine compounds.
Project description:Tissue type plasminogen activator (tPA) can induce neuronal apoptosis, disrupt the blood-brain barrier (BBB), and promote dilation of the cerebral vasculature. The timing, sequence and contributions of these and other deleterious effects of tPA and their contribution to post-ischemic brain damage after stroke, have not been fully elucidated. To dissociate the effects of tPA on BBB permeability, cerebral vasodilation and protease-dependent pathways, we developed several tPA mutants and PAI-1 derived peptides constructed by computerized homology modeling of tPA. Our data show that intravenous administration of human tPA to rats increases BBB permeability through a non-catalytic process that is associated with reversible neurotoxicity, brain damage, mortality and contributes significantly to its brief therapeutic window. Furthermore, our data show that inhibiting the effect of tPA on BBB function without affecting its catalytic activity, improves outcome and significantly extends its therapeutic window in mechanical as well as in thromboembolic models of stroke.
Project description:The plasminogen activation (PA) system is best known for its role in fibrinolysis. However, it has also been shown to regulate many nonfibrinolytic functions in the central nervous system (CNS). In particular, tissue-type plasminogen activator (tPA) is reported to have pleiotropic activities in the CNS, regulating events such as neuronal plasticity, excitotoxicity, and cerebrovascular barrier integrity, whereas urokinase-type plasminogen activator is mainly associated with tissue remodeling and cell migration. It has been suggested that the role tPA plays in controlling barrier integrity may provide a unifying mechanism for the reported diverse, and often opposing, functions ascribed to tPA in the CNS. Here we will review the possibility that the pleiotropic effects reported for tPA in physiologic and pathologic processes in the CNS may be a consequence of its role in the neurovascular unit in regulation of cerebrovascular responses and subsequently parenchymal homeostasis. We propose that this might offer an explanation for the ongoing debate regarding the neurotoxic versus neuroprotective roles of tPA.
Project description:The interaction of bleomycin with a kinetoplast DNA fragment has been examined using various footprinting techniques. This DNA adopts a bent structure and displays an unusually low gel mobility on account of its phased runs of adenines. The bleomycin-cobalt complex increases the mobility of this DNA fragment, in contrast with other DNAs which show a decreased rate of gel migration, suggesting that the antibiotic removes DNA bending, possibly via an unwinding mechanism. Removal of the bending is confirmed by hydroxy-radical footprinting which produces a more even ladder of bands in the presence of the ligand. Cleavage by bleomycin is at the sequence G-pyrimidine, though not all such sites are affected to the same extent and some cutting is found at GA and GG. DNase I footprinting confirms the antibiotic-binding sites but reveals that some strong cleavage sites do not yield footprints. Bleomycin renders adenines on the 3' side of its cleavage sites (GT, GC and GA) hyper-reactive to diethyl pyrocarbonate.
Project description:Four complementary footprinting and probing techniques utilizing DNAse I, methidiumpropyl EDTA (MPE).FeII, diethyl pyrocarbonate (DEPC) and KMnO4 as DNA-cleaving or DNA-modifying agents have been applied to investigate the sequence-specific binding to DNA of the antitumour antibiotic echinomycin. A 265 bp EcoRI-PvuII DNA restriction fragment excised from plasmid pBS was used as a substrate. Six regions of protection against DNAase I cleavage were located on the 265-mer: three sites encompass the sequences 5'-TCGA or 5'-GCGT and the three others contain 5'-GpG (CpC) dinucleotide sequences where the inhibition of DNAase I cutting by echinomycin is less pronounced. In contrast, MPE.FeII cleavage allows identification of only three echinomycin-binding sites on the 265-mer: two sites contain the sequence 5'-TCGA and one encompasses the sequence 5'-ACCA. Cleavage of DNA by MPE.FeII in the presence of echinomycin remains practically unaffected at the sequence 5'-GCGT, despite its identification by DNAase I as a strong site for binding the antibiotic, as well as at the two other sequences containing GpG steps. With both DNAase I and MPE.FeII, enhanced DNA cleavage is evident at AT-rich sequences in the presence of echinomycin. Enhanced reactivity towards KMnO4 and DEPC provides clear evidence for sequence-dependent conformational changes in DNA induced by the antibiotic. The experiments reveal that KMnO4 reacts most strongly with thymines located around, but not necessarily adjacent to, an echinomycin-binding site, whereas the carbethoxylation reactions caused by DEPC occur primarily at the adenine residues lying immediately 5' or 3' to the dinucleotide that denotes an echinomycin-binding site. The results reported here demonstrate that DEPC and KMnO4 serve as sensitive probes for different states of the DNA helix. It seems that the reaction with KMnO4 involves transient unstacking events, whereas the carbethoxylation reaction of DEPC requires larger-scale helix opening.
Project description:The counterion distribution around a DNA dodecamer (5'-CGCGAATTCGCG-3') is analyzed using both standard and novel techniques based on state of the art molecular dynamics simulations. Specifically, we have explored the population of Na(+) in the minor groove of DNA duplex, and whether or not a string of Na(+) can replace the spine of hydration in the narrow AATT minor groove. The results suggest that the insertion of Na(+) in the minor groove is a very rare event, but that when once the ion finds specific sites deep inside the groove it can reside there for very long periods of time. According to our simulation the presence of Na(+) inside the groove does not have a dramatic influence in the structure or dynamics of the duplex DNA. The ability of current MD simulations to obtain equilibrated pictures of the counterion atmosphere around DNA is critically discussed.