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1.
B R Rudolph  D A Case 《Biopolymers》1989,28(4):851-871
Vibrational normal mode calculations are presented for a DNA hexanucleoside pentaphosphate, d(CpGpCpGpCpG)2, and for its complex with the cationic intercalator ethidium. Two intercalation sites are modeled that differ in DNA backbone torsion angles. Normal mode frequencies for the DNA fragment itself are significantly lower than those reported earlier using different force fields, but an analysis of "effective" frequencies suggests that somewhat higher frequencies are more appropriate. Intercalation leads to significant lowering of mobility for the base pairs adjacent to the drug; in this sequence, the ethidium binding affects the guanosine atoms more strongly than the cytosine atoms. Motions of the bases and the intercalator are analyzed in terms of "twist" about the local helix axis and a "tilt" angle relative to this axis, and the results are compared to fluorescence studies of ethidium-DNA complexes.  相似文献   

2.
Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases.  相似文献   

3.
270 MHz 1H NMR and theoretical studies indicate that the drug lucanthone forms intercalated complexes with the synthetic DNA poly(dA-dT)·poly(dA-dT). In the intercalated complex the long axis of the drug is perpendicular to the helix axis and parallel to the base pair axis, i.e., the long axis is perpendicular to the dyad axis.  相似文献   

4.
Structural properties of the complex formed between genomic DNA and the intercalating drug ethidium bromide (EtBr) have been determined by use of a Raman microscope equipped with near-infrared laser excitation. The polarized spectra, which were obtained from oriented fibers of the EtBr:DNA complex, are interpreted in terms of the relative orientations of the phenanthridinium ring of EtBr and bases of DNA. Quantification of structure parameters of EtBr and DNA in the complex were assessed using Raman tensors obtained from polarized Raman analyses of oriented specimens of EtBr (single crystal) and DNA (hydrated fiber). We find that the phenanthridinium plane is tilted by 35+/-5 degrees from the plane perpendicular to the fiber (DNA helix) axis. Assuming coplanarity of the phenanthridinium ring and its immediate base neighbors at the intercalation site, such bases would have a tilt angle closer to that of A-DNA (20 degrees) than to that of B-DNA (6 degrees). The average base tilt in stretches of DNA between intercalation sites remains that of B-DNA.  相似文献   

5.
RNA substrates which form relatively short helices I and III with hammerhead ribozymes are generally cleaved more rapidly than substrates which create longer binding helices. We speculated that for optimum cleavage rates, one of the helices needed to be relatively weak. To identify this helix, a series of ribozymes and substrates of varying lengths were made such that in the complex, helices I and III consisted of 5 and 10 bp respectively or vice versa. In two independent systems, substrates in the complexes with the shorter helix I and longer helix III were cleaved one to two orders of magnitude more rapidly than those in the complexes with the longer helix I and shorter helix III. Similar results were obtained whether the numbers of base pairs in helices I and III were limited either by the length of the hybridizing arms of the ribozyme or the length of the substrate. The phenomenon was observed for both all-RNA and DNA armed ribozymes. Thus, a relatively short helix I is required for fast cleavage rates in pre-formed hammer-head ribozyme-substrate complexes. When helix III has 10 bp, the optimum length for helix I is approximately 5 bp.  相似文献   

6.
Results of calculations using various empirical potentials suggest that base pair buckling, which commonly occurs in DNA crystal structures, is sufficient to eliminate the steric clash at CpG steps in B-DNA, originating from the base pair propeller twisting. The buckling is formed by an inclination of cytosines while deviations of guanines from a plane perpendicular to the double helix axis are unfavorable. The buckling is accompanied by an increased vertical separation of the base pair centers but the buckled arrangement of base pairs is at least as stable as when the vertical separation is normal and buckle zero. In addition, room is created by the increased vertical separation for the bases to propeller twist as is observed in DNA crystal structures. Further stabilization of base stacking is introduced into the buckled base pair arrangement by roll opening the base pairs into the double helix minor groove. The roll may lead to the double helix bending and liberation of guanines from the strictly perpendicular orientation to the double helix axis. The liberated guanines further contribute to the base pair buckling and stacking improvement. This work also suggests a characteristic very stable DNA structure promoted by nucleotide sequences in which runs of purines follow runs of pyrimidine bases.  相似文献   

7.
When designed to cleave a target RNA in trans, the hammerhead ribozyme contains two antisense flanks which form helix I and helix III by pairing with the complementary target RNA. The sequences forming helix II are contained on the ribozyme strand and represent a major structural component of the hammerhead structure. In the case of an inhibitory 429 nucleotides long trans-ribozyme (2as-Rz12) which was directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1), helix II was not pre-formed in the single-stranded molecule. Thus, major structural changes are necessary before cleavage can occur. To study whether pre-formation of helix II in the non-paired 2as-Rz12 RNA could influence the observed cleavage rate in vitro and its inhibitory activity on HIV-1 replication, we extended the 4 base pair helix II of 2as-Rz12 to 6, 10, 21, and 22 base pairs respectively. Limited RNase cleavage reactions performed in vitro at 37 degrees C and at physiological ion strength indicated that a helix II of the hammerhead domain was pre-formed when its length was at least six base pairs. This modification neither affected the association rate with target RNA nor the cleavage rate in vitro. In contrast to this, extension of helix II led to a significantly decreased inhibition of HIV-1 replication in human cells. Together with the finding of others that shortening of helix II to less than two base pairs reduces the catalytic activity in vitro, this observation indicates that the length of helix II in the naturally occurring RNAs with a hammerhead domain is already close or identical to the optimal length for catalytic activity in vitro and in vivo.  相似文献   

8.
D E Gilbert  J Feigon 《Biochemistry》1991,30(9):2483-2494
The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.  相似文献   

9.
DNA-fiber EPR spectroscopy and its application to studies of the DNA binding orientation and dynamic properties of Cu(II) ions and their complexes with amino acids and peptides are reviewed. Cu(II) ions bind in at least two different binding modes; one mode was mobile while the other mode fixed the orientation of the coordination plane. The hydroxyl groups of L-Ser and L-Thr fixed the coordination plane of their respective Cu(II) complexes parallel to the DNA base pair plane, whereas Cu(II) complexes of Lys and Arg induced several binding modes, depending on the tertiary structure of the DNA and the chirality of the amino acids. Unusually broadened signals observed for the His complex were assigned to a mono-L-His complex stacked stereospecifically along the DNA double helix. In comparison, Cu(II). Xaa-Xaa' -His type complexes oriented in the minor groove with different affinities and extents of randomness depending on the Xaa-Xaa' sequence and the chirality of Xaa or Xaa' while the C-terminal Xaa residues in Cu(II).Arg-Gly-His-Xaa (Xaa=L-Leu or L-Glu) decreased the stereospecificity and the stability of the complexes bound to DNA. In contrast to Xaa-Xaa'- His complexes, the coordination planes of Cu(II).Gly-L-His-Gly and Cu(II).Gly-L-His-L-Lys complexes were found to lie parallel to the DNA-fiber axis. Dinuclear Cu(II).carnosine complexes were also shown to bind to DNA stereospecifically.  相似文献   

10.
S A White  D E Draper 《Biochemistry》1989,28(4):1892-1897
The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.  相似文献   

11.
At low temperature and low salt concentration, both imino proton and 31p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in 31P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.  相似文献   

12.
Comparative 1H NMR and optical studies of the interaction between poly(dA-dT), ethidium bromide (Et) and ethidium dimer (Et2) in 0.7 M NaCl are reported as a function of the temperature. Denaturation of the complexes followed at both polynucleotide and drug levels leads to a biphasic melting process for poly(dA-dT) complexed with ethidium dimer (t1/2 = 75 degrees C; 93 degrees C) but a monophasic one in poly(dA-dT): ethidium bromide complex (t1/2 = 74 degrees C). In both cases drug signals exhibit monophasic thermal dependence (Et = 81 degrees C; Et2 = 95 degrees C). Evidence is presented showing that the ethidium dimer bisintercalates into poly(dA-dT) in high salt, based on the observation that i) dimer and monomer ring protons exhibit similar upfield shifts upon DNA binding, ii) upfield shifts of DNA sugar protons are twice as large with the dimer than with ethidium bromide. Comparison between native DNA fraction and bound drug fraction indicates that ethidium covers, n = 2.5-3 base pairs. The dimer bisintercalates and covers, n = 5.7 base pairs when the helix fraction is high but as the number of available sites decreases the binding mode changes and the drug monointercalates (n = 2.9).  相似文献   

13.
Single crystals of the self-complementary octadeoxyribonucleotide d(GCCCGGGC) have been analysed by X-ray diffraction methods at a resolution of 1.8 A. The tetragonal unit cell of space group P4(3)2(1)2 has dimensions of a = 43.25 A and c = 24.61 A and contains eight strands of the oligonucleotide. The structure was refined by standard crystallographic techniques to an R factor of 17.1% using 1359 3 sigma structure factor observations. Two strands of the oligonucleotide are related by the crystallographic dyad axis to form a DNA helix in the A conformation. The d(GCCCGGGC) helix is characterized by a wide open major groove, a near perpendicular orientation of base pairs to the helix axis and an unusually small average helix twist angle of 31.3 degrees indicating a slightly underwound helix with 11.5 base pairs per turn. Extensive cross-strand stacking between guanine bases at the central cytosine-guanine step is made possible by a number of local conformational adjustments including a fully extended sugar-phosphate backbone of the central guanosine nucleotide.  相似文献   

14.
Abstract

Results of calculations using various empirical potentials suggest that base pair buckling, which commonly occurs in DNA crystal structures, is sufficient to eliminate the steric clash at CpG steps in B-DNA, originating from the base pair propeller twisting. The buckling is formed by an inclination of cytosines while deviations of guanines from a plane perpendicular to the double helix axis are unfavorable. The buckling is accompanied by an increased vertical separation of the base pair centers but the buckled arrangement of base pairs is at least as stable as when the vertical separation is normal and buckle zero. In addition, room is created by the increased vertical separation for the bases to propeller twist as is observed in DNA crystal structures. Further stabilization of base stacking is introduced into the buckled base pair arrangement by roll opening the base pairs into the double helix minor groove. The roll may lead to the double helix bending and liberation of guanines from the strictly perpendicular orientation to the double helix axis. The liberated guanines further contribute to the base pair buckling and stacking improvement. This work also suggests a characteristic very stable DNA structure promoted by nucleotide sequences in which runs of purines follow runs of pyrimidine bases.  相似文献   

15.
Interaction of Δ,Δ- and Λ,Λ-bis-Ru(II) complexes with native DNA was investigated by isotropic absorption and polarized spectroscopy including circular and linear dichroism (CD and LD). Despite the steric hindrance originating from its four bulky phenanthroline ligands at both ends of the molecule, this molecule rapidly intercalates between DNA base pairs. Intercalation was judged by large hypochromism and red shift in the UV-visible absorption spectra in the absorption region of the bridging moiety as well as in the metal-to-ligand-charge transfer absorption region. Further support for the intercalation is found in the fact that the magnitude of negative reduced LD signal in the absorption region of the bridging moiety was comparable to that of the DNA absorption region, indicating that the bridge connecting the two Ru(II) complexes is nearly parallel to the DNA base planes. No difference in the binding mode between the two enantiomers was observed. In the presence of either bis-Ru(II) complex, ethidium bromide, a classical intercalator, can intercalate into the empty sites but was not able to replace the Ru(II) complexes. Near the saturation, ground state interaction between ethidium and bis-Ru(II) complex was evident by LD.  相似文献   

16.
Magnetic ordering of DNA liquid crystals   总被引:2,自引:0,他引:2  
R Brandes  D R Kearns 《Biochemistry》1986,25(20):5890-5895
Sonicated calf thymus DNA with an average length of approximately 100 base pairs has been found to form a cholesteric liquid crystal at a concentration of approximately 250 mg of DNA/mL of solution. Immediately after preparation, small ordered domains of a few micrometers are formed, resulting in an opaque solution. This liquid crystal can readily be oriented in the magnetic field of an NMR magnet, resulting in a clear birefringent phase. The DNA molecules align with their helix axes perpendicular to the field so that the cholesteric pitch axis was parallel with the field. A pitch length of approximately 2.5 microns for the cholesteric phase was determined both from optical measurements (optical light rotation) and from NMR measurements (solvent diffusion). The observation that DNA molecules can be magnetically oriented opens up new possibilities for studying the structure and dynamics of the aligned DNA molecules.  相似文献   

17.
The binding positions and relative minimum binding energies are calculated for complexes of 9-aminoacridine, proflavine, N-methylphenanthridinium, and ethidium in theoretically determined intercalation sites in B-DNA (sites I and II) and in unconstrained dimer-duplex sites. The selection of site I in B-DNA by these compounds agrees with the theoretical interpretation of studies of unwinding angles in closed circular DNA in all cases but ethidium, which is predicted to select site II. The most stable binding positions of the acridines and ethidium in unconstrained dimer-duplex units agree with experimental results of intercalation complexes of dinucleoside monophosphate units. Base-pair specificity for Watson-Crick pairing is examined. The energy of an intercalation complex is partitioned into ΔE23, the energy required to open base pairs BP2 and BP3 in B-DNA to a site, and ΔEIn, the energy change when a free molecular intercalates. ΔE23 depends strongly on the base-pair sequence, whereas ΔEIn for the four molecules studied does not. The three most stable sequences contain (pyrimidine)p(purine) units, and this provides a rationale for the exclusive formation of crystals of intercalation complexes with these units. In spite of this selectivity, the distribution of G?C and A?T base pairs is equal for these three units and persists as the more unstable sequences are included. Therefore, specificity arises from the interaction between the base pairs and the 2′-deoxyribose 5′-monophosphate backbone for the opening of B-DNA to an intercalation site and not from the interaction between the chromophore and the DNA.  相似文献   

18.
Kabanov AV  Komarov VM  Perez V 《Biofizika》2005,50(3):434-443
On the basis of the idea of the intrinsic polymorphism of Watson-Crick base pairing in DNA structure, the process of accumulation of the large electric dipole moment in model spiral stacks of canonical non-planar AT and GC pairs was analyzed using the quantum-chemistry methods. The dependence of the value and orientation of electrical dipole moment of a double helix on spiral length, geometry of base H-pairing, and the bending of the major axis of the helix were considered.  相似文献   

19.
Circular dichroism was applied to the analysis of drug-DNA associations. With the octanucleotide d(TGACGTCA) (octanucleotide I), which is the cAMP-responsive element (CRE) in gene promoters and its reverse d(ACTGCAGT) (octanucleotide II), it was demonstrated that the anticancer polyaromatic agent celiptium intercalates into DNA base pairs with its long direction perpendicular to both the DNA-helix axis and the base-pair long axis and induces larger conformational changes in the CpG-containing octanucleotide I CRE than in its reverse-sequence octanucleotide II. It was concluded that CD is a powerful and sensitive technique to discriminate between drug-binding modes of DNA, to define the geometry of the chromophore inserted into base pairs and, finally, to measure sequence-dependent conformational changes induced by intercalation in DNA. We anticipate that these studies will contribute to a better understanding of the molecular bases that underlie the mechanism of action of those cytotoxic drugs which interfere with the DNA-nuclear-protein recognition.  相似文献   

20.
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