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R H Shafer

Publications and source records attributed to R H Shafer.

At least 37 records · Page 2Linked to original sources
PubMed1993

Differential effects of spermine and its analogues on the structures of polynucleotides complexed with ethidium bromide.

The interactions of spermine and polyamine analogues with synthetic polynucleotides of various base sequences complexed with ethidium bromide (EB) were investigated using measurements of fluorescence intensity and steady-state fluorescence polarization. Spermine and polyamine analogues displaced some but not all of the EB bound to poly(dA-dT).poly(dA-dT) or poly(dG-dC).poly(dG-dC), suggesting that polyamines may stabilize these polynucleotides in a conformation with reduced affinity for EB. Modifications of the aliphatic backbone of spermine have pronounced effects on its ability to displace EB from poly(dA-dT).poly(dA-dT) but not from poly-(dG-dC).poly(dG-dC). Spermine and some but not all of the polyamine analogues caused fluorescence depolarization when they interacted with the complex of EB and poly(dA-dT).poly-(dA-dT). Neither spermine nor any of the analogues, however, induced fluorescence depolarization in the complex of EB with poly(dG-dC).poly(dG-dC) or poly(dA).poly(dT). This suggests that spermine and some spermine analogues induce structural changes specific to alternating A-T sequences.

Ethidium↗
PubMed1993

Differential binding of the enantiomers of chloroquine and quinacrine to polynucleotides: implications for stereoselective metabolism.

Interaction of the antimalarial drugs quinacrine and chloroquine with DNA has been studied extensively in order to understand the origin of their biological activity. These studies have shown that they bind to DNA through an intercalative mode and show little sequence specificity. All previous experiments were carried out using the racemic form of these drugs. We have investigated the binding of the enantiomeric forms of quinacrine and chloroquine to synthetic polynucleotides poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly (dG-dC), and found interesting differences in their binding parameters. Quinacrine enantiomers have a much higher binding affinity for the two polynucleotides compared to those of chloroquine. The negative enantiomers were found to have higher binding affinity than the positive ones. The binding constant for the binding of quinacrine(-) to poly(dG-dC).poly(dG-dC) was found to be about 3 times that of quinacrine(+). The differences in these binding affinities were further confirmed by equilibrium dialysis of the complexes of the polynucleotides with the racemic form of the drugs, which resulted in the enrichment of the dialysate with the positive enantiomer. CD spectra of the enantiomers and their polynucleotide complexes are reported. Changes in the fluorescence properties of quinacrine in the presence of the two polynucleotides are also described. Biological implications of these findings are discussed.

Chloroquine↗
PubMed1992

Quadruplex structure of d(G3T4G3) stabilized by K+ or Na+ is an asymmetric hairpin dimer.

The ends of chromosomes contain repeats of guanine-rich sequences that can assume highly compact conformations and are presumed necessary for their biological role in chromosomal stabilization and association. We have investigated the conformational behavior of d(G3T4G3) as a function of the addition of either KCl or NaCl, in the concentration range of 50-200 mM, by using a spectrum of physical techniques and conclude that these salts induce a quadruplex species composed of two strands, each in a hairpin conformation. When salt is added, a large positive signal appears near 290 nm in the CD spectra. UV thermal denaturation curves show a single concentration-dependent transition and provide data for quantitating the thermodynamics of quadruplex formation. In electrophoresis experiments, the quadruplex structure migrates as a single species and more rapidly than the unstructured single strand. NMR spectra in the presence of KCl or NaCl indicate that the structure formed is asymmetric. Equilibrium ultracentrifugation studies confirm that these quadruplexes are composed of two strands of d(G3T4G3). Possible models for this structure are discussed.

Base Sequence↗
PubMed1992

The octamer motif in immunoglobulin genes: extraction of structural constraints from two-dimensional NMR studies.

Phase-sensitive two-dimensional nuclear Overhauser enhancement (2D NOE) and double-quantum-filtered correlated (2QF-COSY) spectra were recorded at 500 MHz for the DNA duplex d(CATTTGCATC).d(GATGCAAATG), which contains the octamer element of immunoglobulin genes. Exchangeable and nonexchangeable proton resonances including those of the H5' and H5" protons were assigned. Overall, the decamer duplex adopts a B-type DNA conformation. Scalar coupling constants for the sugar protons were determined by quantitative simulations of 2QF-COSY cross-peaks. These couplings are consistent with a two-state dynamic equilibrium between a minor N- and a major S-type conformer for all residues. The pseudorotation phase angle P of the major conformer is in the range 117-135 degrees for nonterminal pyrimidine nucleotides and 153-162 degrees for nonterminal purine nucleotides. Except for the terminal residues, the minor conformer comprises less than 25% of the population. Distance constraints obtained by a complete relaxation matrix analysis of the 2D NOE intensities with the MARDIGRAS algorithm confirm the dependence of the sugar pucker on pyrimidine and purine bases. Averaging by fast local motions has at most small effects on the NOE-derived interproton distances.

Animals↗
PubMed1992

H-NMR studies on d(GCTTAAGC)2 and its complex with berenil.

Two-dimensional (2D) 1H-NMR spectroscopy has been used to analyze the structure of d(GCTTAAGC)2 and its interaction with berenil in solution. Nuclear Overhauser enhancement connectivities enabled sequential assignments of nearly all proton resonances in the self-complementary octamer duplex and demonstrated that the oligonucleotide is primarily in a B-type conformation. No major conformational changes were observed by the addition of berenil, but proton resonances of the two adenosine nucleotides shifted substantially. Intermolecular nuclear Overhauser effects between berenil and the DNA duplex revealed that the drug binds via the minor groove of d(GCTTAAGC)2 in the A.T-base-pair region. At 18 degrees C the twofold symmetry of the duplex is preserved on berenil binding. However, strongly shifted proton resonances broadened significantly. A model is proposed for the berenil-d(GCTTAAGC)2 complex involving fast exchange of berenil between two equivalent symmetry-related binding sites, which span the 5'-TAA-3' region and are asymmetrically disposed with respect to the dyad axis of the duplex. These results are compared with previous studies on the berenil-d(GCAATTGC)2 complex.

Base Sequence↗
PubMed1991

NMR investigation of the interaction of mithramycin A with d(ACCCGGGT)2.

The binding of mithramycin A to d(ACCCGGGT)2 has been investigated by one- and two-dimensional 1H NMR spectroscopy. Titration of the drug into the octamer solution results in loss of the oligonucleotide C2 symmetry at stoichiometric ratios less than 4 drug molecules per duplex. However, at a ratio of 4:1 (drug/duplex), the C2 symmetry of the oligonucleotide is restored. From these data it is evident that more than one complex forms at ratios less than 4:1 while only one complex predominates at the ratio 4:1. This is the first report of a DNA octamer which binds 4 large drug molecules. These results are compared to those we have recently reported for mithramycin binding to d(ATGCAT)2, where only a single, bound complex is observed, with a stoichiometry of 2:1.

Base Sequence↗
PubMed1991

Structure, stability, and thermodynamics of a short intermolecular purine-purine-pyrimidine triple helix.

We have investigated the structure and physical chemistry of the d(C3T4C3).2[d(G3A4G3)] triple helix by polyacrylamide gel electrophoresis (PAGE), 1H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl2 at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur.pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the purine strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 degrees C, depending on the DNA concentration. The free energy of triplex formation (-26.0 +/- 0.5 kcal/mol) is approximately twice that of duplex formation (-12.6 +/- 0.7 kcal/mol), suggesting that the overall stability of the pur.pur base pairs is similar to that of the W-C base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1991

1H and 31P nuclear magnetic resonance studies of spermine binding to the Z-DNA form of d(m5CGm5CGm5CG)2. Evidence for decreased spermine mobility.

The binding of spermine to the d(m5CGm5CGm5CG) duplex has been studied by proton and phosphorus nuclear magnetic resonance techniques in order to investigate the mobility and nature of spermine bound to the resulting Z-DNA complex. A characterization of the B to Z transition as a function of increasing spermine concentration demonstrated doubling of the non-exchangeable proton and the phosphorus peaks at a ratio of about 1:1 (spermine/duplex) and a re-simplification of the spectrum at 2:1 (spermine/duplex) where about 90% or the DNA was fully converted into the Z-form. However, some of the Z-DNA proton chemical shifts differed between the 1:1 and 2:1 titration points. Since these differences involved primarily the exchangeable terminal imino and amino protons, they could result from end effects. Discrepancies were generally not observed with the non-terminal proton shifts nor with the phosphorus shifts. These proton and phosphorus chemical shift changes are fully consistent with a B to Z transition. Complexed spermine peaks appear about 0.1 parts per million upfield from the uncomplexed form. The spermine and both the B and Z-DNA hexamer signals are noticeably broadened at the 1:1 ratio but the remaining signals re-sharpen at the 2:1 ratio. Both one-dimensional and two-dimensional studies revealed negative nuclear Overhauser effect (NOE) contacts between each spermine proton. Therefore, spermine has a longer correlation time than that observed for unbounded spermine. These results are contrasted with the positive NOE contacts observed for the B-DNA-spermine complexes reported by Wemmer et al. using the dodecamer d(CGCGAATTCGCG)2 and reported here using the hexamer d(ATGCAT)2. While the mobility of spermine in the Z-DNA complex is significantly less than that of the B-DNA complex, no clear evidence of intermolecular spermine-DNA proton NOE contacts is observed.

Base Sequence↗
PubMed1991

Binding of ethidium bromide to a DNA triple helix. Evidence for intercalation.

The interaction of ethidium, a DNA intercalator, with the poly(dA).poly(dT) duplex and the poly (dA).2poly(dT) triplex has been investigated by a variety of spectrophotometric and hydrodynamic techniques. The fluorescence of ethidium is increased when either the duplex or triplex form is present. Binding constants, determined from absorbance measurements, indicate that binding to the triple helical form is substantially stronger than to the duplex, with a larger binding site size (2.8 base triplets compared to 2.4 base pairs). Furthermore, while binding to poly(dA).poly(dT) shows strong positive cooperativity, binding to the triplex is noncooperative. Thermal denaturation experiments demonstrate that ethidium stabilizes the triple helix. Binding to either form induces a weak circular dichroism band in the visible wavelength region, while in the region around 310 nm, there is a band that is strongly dependent on the degree of saturation of the duplex, and which is positive for the duplex but negative for the triplex. Both fluorescence energy transfer and quenching studies provide evidence of intercalation of ethidium in both duplex and triplex complexes. Binding of ethidium leads to an initial decrease in viscosity for both the duplex and triplex structures, followed by an increase, which is greater for the duplex. Taken together, these results strongly suggest that ethidium binds to the poly (dA).2poly(dT) triple helix via an intercalative mechanism.

Binding Sites↗
PubMed1990

Thermodynamics of triple helix formation: spectrophotometric studies on the d(A)10.2d(T)10 and d(C+3T4C+3).d(G3A4G3).d(C3T4C3) triple helices.

We have stabilized the d(A)10.2d(T)10 and d(C+LT4C+3).d(G3A4G3).d(C3T4C3) triple helices with either NaCl or MgCl2 at pH 5.5. UV mixing curves demonstrate a 1:2 stoichiometry of purine to pyrimidine strands under the appropriate conditions of pH and ionic strength. Circular dichroic titrations suggest a possible sequence-independent spectral signature for triplex formation. Thermal denaturation profiles indicate the initial loss of the third strand followed by dissociation of the underlying duplex with increasing temperature. Depending on the base sequence and ionic conditions, the binding affinity of the third strand for the duplex at 25 degrees C is two to five orders of magnitude lower than that of the two strands forming the duplex. Thermodynamic parameters for triplex formation were determined for both sequences in the presence of 50 mM MgCl2 and/or 2.0 M NaCl. Hoogsteen base pairs are 0.22-0.64 kcal/mole less stable than Watson-Crick base pairs, depending on ionic conditions and base composition. C+.G and T.A Hoogsteen base pairs appear to have similar stability in the presence of Mg2+ ions at low pH.

Circular Dichroism↗
PubMed1990

NMR investigation of mithramycin A binding to d(ATGCAT)2: a comparative study with chromomycin A3.

The binding of mithramycin A to the d(A1T2G3C4A5T6) duplex was investigated by 1H NMR and found to be similar to that of its analogue chromomycin A3. In the presence of Mg2+, mithramycin binds strongly to d(ATGCAT)2. On the basis of the two-dimensional NOESY spectrum, the complex formed possesses C2 symmetry at a stoichiometry of two drugs per duplex (2:1) and is in slow chemical exchange on the NMR time scale. NOESY experiments reveal contacts from the E-pyranose of mithramycin to the terminal and nonterminal adenine H2 proton of DNA and from the drug hydroxyl proton to both G3NH2 protons, C4H1' proton, and A5H1' proton. These data place the drug chromophore and E pyranose on the minor groove side of d(ATGCAT)2. NOE contacts from the A-, B-, C-, and D-pyranoses of mithramycin to several deoxyribose protons suggest that the A- and B-rings are oriented along the sugar-phosphate backbone of G3-C4, while the C- and D-rings are located along the sugar-phosphate backbone of A5-T6. These drug-DNA contacts are very similar to those found for chromomycin binding to d(ATGCAT)2. Unlike chromomycin, the NOESY spectrum of mithramycin at the molar ratio of one drug per duplex reveals several chemical exchange cross-peaks corresponding to the drug-free and drug-bound proton resonances. From the intensity of these cross-peaks and the corresponding diagonal peaks, the off-rate constant was estimated to be 0.4 s-1. These data suggest that the exchange rate of mithramycin binding to d(ATGCAT)2 is faster than that of chromomycin.

Base Sequence↗
PubMed1990

Structural analysis of d(GCAATTGC)2 and its complex with berenil by nuclear magnetic resonance spectroscopy.

The structures of d(GCAATTGC)2 and its complex with berenil in solution were analyzed by two-dimensional 1H NMR spectroscopy. Intra- and internucleotide nuclear Overhauser effect (NOE) connectivities demonstrate that the octanucleotide duplex is primarily in the B conformation. Binding with berenil stabilizes the duplex with respect to thermal denaturation by about 10 degrees C, based on the appearance of the imino proton signals. The berenil-d(GCAATTGC)2 system is in fast exchange on the NMR time scale. The two-dimensional NMR data reveal that berenil binds in the minor groove of d(GCAATTGC)2. The aromatic drug protons are placed within 5 A of the H2 proton of both adenines, the H1', H5', and H5" of both thymidines, and the H4', H5', and H5" of the internal guanosine. The amidine protons on berenil are also close to the H2 proton of both adenines. The duplex retains an overall B conformation in the complex with berenil. At 18 degrees C, NOE contacts at longer mixing times indicate the presence of end-to-end association both in the duplex alone and also in its complex with berenil. These intermolecular contacts either vanished or diminished substantially at 45 degrees C. Two molecular models are proposed for the berenil-(GCAATTGC)2 complex; one has hydrogen bonds between the berenil amidine protons and the carbonyl oxygen, O2, of the external thymines, and the other has hydrogen bonds between the drug amidine protons and the purine nitrogen, N3, of the internal adenines. Quantitative analysis of the NOE data favors the second model.

Amidines↗
PubMed1990

NMR studies of the interaction of chromomycin A3 with small DNA duplexes. Binding to GC-containing sequences.

The interaction of chromomycin A3 with the oligodeoxyribonucleotides 1, d(ATGCAT), 2, d(ATCGAT), 3, d(TATGCATA), and 4, d(ATAGCTAT), has been investigated by 1H and 31P NMR. In the presence of Mg2+, chromomycin binds strongly to the three GC-containing oligomers 1, 3, and 4 but not to the CG-containing oligomer 2. The proton chemical shift changes for 1 and 3 are similar, and these DNA duplexes appear to bind with a stoichiometry of 2 drugs:1 Mg2+:1 duplex. The same stoichiometry of 2 drugs:1 duplex is confirmed with 4; however, proton chemical shift changes differ. An overall C2 symmetry is exhibited by the drug complex with 1, 3, and 4. At a molar ratio of 2.0 (drugs:duplex), no free DNA proton NMR signals remain. Two-dimensional nuclear Overhauser exchange spectroscopy (NOESY) of the saturated chromomycin complex with 1 and 3 positions both chromomycinone hydroxyls and the E carbohydrates in the minor groove and provides evidence suggesting that the B carbohydrates lie on the major-groove side. This is supported by several dipolar coupling cross-peaks between the drug and the DNA duplex. Drug-induced conformational changes in duplex 1 are evaluated over a range of NOESY mixing times and found to possess some characteristics of both B-DNA and A-DNA, where the minor groove is wider and shallower. A widening of the minor groove is essential for the DNA duplex to accommodate two drug molecules. This current minor-groove model is a substantial revision of our earlier major-groove model [Keniry, M.A., Brown, S.C., Berman, E., & Shafer, R.H. (1987) Biochemistry 26, 1058-1067] and is in agreement with the model recently proposed by Gao and Patel [Gao, X., & Patel, D. J. (1989a) Biochemistry 28, 751-762].

Base Composition↗
PubMed1990

Structural analysis of the (dA)10.2(dT)10 triple helix.

The existence of DNA triple helices in vitro has been known for some time. Recent evidence suggesting that DNA triplexes exist in vivo and showing their potential for chemotherapeutic applications has renewed interest in these triple-strand conformations. However, little structural information is currently known about these unusual nucleic acid forms. We have induced and stabilized triple-helical (dA)10.2(dT)10 with MgCl2 at neutral pH. UV mixing curves demonstrate a 1:2 (dA)10 to (dT)10 stoichiometry at suitable MgCl2 concentrations. Thermal denaturation profiles establish a melting mechanism characterized by the initial loss of the third strand, followed by dissociation of the remaining duplex. The circular dichroic spectrum of the triplex form is distinct from that of a duplex equimolar in (dA)10. NMR studies show that magnesium-induced triplex formation is accompanied by an upfield shift of several imino proton resonances present before stabilization of the triplex form with MgCl2 and the induction of new upfield imino proton resonances. Nuclear Overhauser effect spectroscopy measurements on both undeuterated and C8--H-deuterated (dA)10.2(dT)10 triplexes demonstrate dipolar contacts between resolvable imino proteins and both adenine C8--H and C2--H aromatic protons. Hence, MgCl2 stabilizes a triplex structure in which thymine N3--H imino protons are involved in both Watson-Crick and Hoogsteen base pairing.

Base Composition↗
PubMed1989

Binding of actinomycin D to [d(ATCGAT)]2: NMR evidence of multiple complexes.

Actinomycin D (actD) binds to the oligonucleotide [d(ATCGAT)]2 with a hypochromatic and red-shifted visible absorbance band compared to free drug and a CD spectrum with double negative bands at 460 and 385 nm. These spectral features are similar to those of the actD-[d(ATGCAT)]2 complex, while actD-[d(AT)5]2 gives spectra similar to those of free drug. Upon dilution or raising the temperature, the spectral characteristics accompanying complex formation disappear in the actD-[(ATCGAT)]2 sample but remain in the actD-[d(ATGCAT)]2 complex under the same experimental conditions. These results suggest that (a) sequence-specific binding of actD occurs with [d(ATCGAT)]2 but not with [d(AT)5]2, (b) the binding is not as strong as with [d(ATGCAT)]2, and (c) actD binds [d(ATCGAT)]2 with the same mechanism as it binds [d(ATGCAT)]2, i.e., by intercalation. From NMR spectra of the actD-[d(ATCGAT)]2 complex, three types of signals can be detected below 20 degrees C, one major and two minor ones. At higher temperatures, exchange between the two minor ones becomes fast enough that only one type of minor signal was seen. Partial resonance assignments were made by using 2D nuclear Overhauser effect (NOE) and 2D homonuclear Hartmann-Hahn (HOHAHA) experiments. Proton chemical shift changes of the major complex are consistent with actD chromophore ring intercalation between hexamer base pairs. Data from NOE-detected dipolar interactions between actD and [d(ATCGAT)]2 protons were interpreted in terms of a major complex with the actD chromophore ring system intercalated at the CG position and minor complexes with the drug intercalated off center at the GA positions.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1988

Solution conformation of the antitumor antibiotic chromomycin A3 determined by two-dimensional NMR spectroscopy.

A conformational analysis and a complete assignment of the nonexchangeable proton resonances of chromomycin A3, dechromose-A chromomycin A3, and deacetylchromose-B chromomycin A3 were carried out in organic solvents. The resulting conformation in methanol has the three side chains of chromomycin A3 fully extended, away from one another and from the aglycon. In dichloromethane on the other hand, the drug was shown to adopt a highly compact conformation in which most of the 26 oxygen atoms in the molecule point out toward the solvent. The two carbohydrate side chains extend parallel to each other on the same side of the aglycon. Two intramolecular nuclear Overhauser enhancement contacts have been observed between different sugar units on these side chains, indicating close proximity for these moieties. In addition, the aliphatic side chain is folded toward the aglycon, parallel to the two oligosaccharide side chains. The overall conformation has a wedge-like shape with the two phenoxy groups exposed at the pointed edge. The presence of some exchange cross-peaks in the NOESY spectra suggests the presence of intramolecular hydrogen bonds that probably help to maintain the compact conformation. The derivatives of chromomycin A3 have qualitatively similar conformations, though their respective conformations are not as compact as the parent drug. The significance of these results is discussed in terms of a model of chromomycin A3 binding to DNA in the major groove.

Chromomycin A3↗
PubMed1988

Chromomycin A3 binds to left-handed poly(dG-m5dC).

The interaction of chromomycin A3 (an antitumor antibiotic) with right-handed and left-handed polynucleotides has been studied by absorbance, fluorescence, circular dichroism, 31P-NMR and 1H-NMR techniques. Binding to either the B form of poly(dG-dC) or the Z form of poly(dG-m5dC) shifts the absorbance maximum to higher wavelength and enhances the fluorescence of the drug. Circular dichroic spectra of solutions containing various concentrations of chromomycin A3 and fixed concentrations of either B or Z polynucleotides show well defined isoelliptic points at similar wavelengths. At the isoelliptic point, the drug complex with B DNA exhibits positive ellipticity while with Z DNA it exhibits negative ellipticity. 31P-NMR spectra of the chromomycin A3 complex with the Z form of poly(dG-m5dC) demonstrate that the Z conformation is retained in the drug complex up to one molecule drug/four base pairs. At Mg2+ concentrations lower than that necessary to stabilize the left-handed conformation of poly(dG-m5dC) alone, 31P analysis shows that chromomycin A3 can bind simultaneously to both the B and Z conformations of poly(dG-m5dC), with no effect on the B-Z equilibrium. These data demonstrate that chromomycin A3 binds to left-handed poly(dG-m5dC) with retention of the left-handed conformation up to saturating drug concentrations.

Binding Sites↗