PubMed Health⌕ Search

Biomedical subjects

R B Macgregor

Publications and source records attributed to R B Macgregor.

At least 19 recordsLinked to original sources
PubMed2003

Cellular uptake and metabolism of DNA frayed wires.

DNA frayed wires are a novel, multistranded form of DNA that arises from interactions between single-stranded oligodeoxyribonucleotides with the general sequence d(N(x)G(y)) or d(G(y)N(x)), where y > 10 and x > 5. Frayed wires exhibit greater stability with respect to thermal and chemical denaturation than single- or double-stranded DNA molecules and, thus, may have potential usefulness for DNA drug delivery. However, the stability and uptake of frayed wires have not been investigated in biological systems. Our objective was to examine the cellular uptake and stability of frayed wires in cultured hepatic cells. In these studies, the parent oligonucleotide d(A(15)G(15)) was used to form DNA frayed wires (DNA(FW)) while a random 30-mer oligonucleotide was used as the control nonaggregated DNA (DNA(SS)). Uptake and metabolism studies of DNA(FW) were performed in cultured human hepatoma, HepG2 cells and compared to DNA(SS). Our results indicate that DNA(FW) are not cytotoxic and that their intracellular uptake in HepG2 cells is 2-3.5-fold greater than that of DNA(SS) within the first 2 h (p < 0.05). Similarly, nuclear localization of DNA(FW) is 10-13-fold higher than that of DNA(SS) (p < 0.05). As both internalized and extracellular DNA(FW) appear to be more stable in vitro than DNA(SS), the enhanced uptake may be due to either increased stability or enhanced intracellular transport. These studies also indicate that uptake of DNA(FW) likely occurs via active processes such as receptor-mediated endocytosis similar to mechanisms which have been proposed for DNA(SS). The internalization pathways of DNA(FW) may differ somewhat from that of DNA(SS) insofar as chloroquine does not appear to alter DNA(FW) uptake and degradation, as is the case with DNA(SS).

Adenosine Triphosphate↗
PubMed2001

On the stability of double stranded nucleic acids.

We present the first pressure-versus-temperature phase diagram for the helix-to-coil transition of double stranded nucleic acids. The thermodynamic stability of a nucleic acid duplex is a complex function of temperature and pressure and strongly depends on the denaturation temperature, T(M), of the duplex at atmospheric pressure. Depending upon T(M), pressure, and temperature, the phase diagram shows that pressure may stabilize, destabilize, or have no effect on the conformational state of DNA. To verify the phase diagram, we have conducted high-pressure UV melting experiments on poly(dIdC)poly(dIdC), a DNA duplex, poly(rA)poly(rU), an RNA duplex, and poly(dA)poly(rU), a DNA/RNA hybrid duplex. The T(M) values of these duplexes have been modulated by altering the solution ionic strength. Significantly, at low salt, these three duplexes have helix-to-coil transition temperatures of 50 degrees C or less. In agreement with the derived phase diagram, we found that the polymeric duplexes were destabilized by pressure if the T(M) is < approximately 50 degrees C. However, these duplexes were stabilized by pressure if the T(M) is > approximately 50 degrees C. The DNA/RNA hybrid duplex, poly(dA)poly(rU), with a T(M) of 31 degrees C in 20 mM NaCl undergoes a pressure-induced helix-to-coil transition at room temperature. This is the first report of pressure-induced denaturation of a nucleic acid duplex and provides new insights into the molecular forces stabilizing these structures.

DNA↗
PubMed2000

Formation and structural determinants of multi-stranded guanine-rich DNA complexes.

We have investigated the complexes formed by oligonucleotides with the general sequence d(T15,Gn), where n = 4-15. Two distinct classes of structures are formed, namely, the four-stranded tetraplex and frayed wires. Frayed wires differ from four-stranded tetraplexes in both strand association stoichiometry and the ability of dimethyl sulfate to methylate the N7 position of guanine. Thus, it appears that these two guanine-rich multistranded assemblies are stabilised by different guanine-guanine interactions. The number of contiguous guanine residues determines which of the complexes is favoured. Based on the stoichiometry of the associated species and the accessibility of the N7 position of guanine to methylation we have found that oligonucleotides with smaller number of contiguous guanines; n = 5-8, form primarily four-stranded tetraplex. Oligonucleotides with larger numbers of contiguous guanines adapt primarily the frayed wire structure. The stability of the complexes formed by this series of oligonucleotides is determined by the number and arrangement of the guanines within the sequences. We propose that the formation of the two types of complex proceed by a parallel reaction pathways that may share common intermediates.

Base Sequence↗
PubMed2000

Thermal activation of DNA frayed wire formation.

Dynamic light scattering has been used to study the formation of stable multistranded DNA complexes called frayed wires. DNA frayed wires arise from the indefinite self-association of oligonucleotides with long terminal tracks of guanines, e.g. d(A15G15). The complexes are stabilized via guanine-guanine interactions resulting in the formation of a guanine stem. Non-guanine portions of the oligonucleotide are disposed away from the stem and form single stranded arms. The indefinite nature of the self-association of these oligonucleotides leads to a distribution of aggregate molecular weights. The distribution arises from aggregated species that differ from one another by the number of self-associated oligonucleotides. In light-scattering experiments, the autocorrelation functions collected for frayed wires are bimodal. The slow mode, often observed for samples containing DNA and other polyelectrolytes, has been ascribed to the formation of large unspecific aggregates due to electrostatic or other long-range interactions. We attribute the fast mode to the translational diffusion of the polydisperse population in the frayed wire sample. We use the mean of the fast mode to characterize the growth of the frayed wires. Consistent with the gel electrophoresis studies, the aggregation of frayed wires is promoted by the presence of magnesium ions and incubation at high temperature. The rate of aggregate formation increases with temperature, indicating the positive activation energy for the reaction. We propose an energy diagram for the formation/disruption of frayed wires revealing the catalytic-like role of the complementary strand in the denaturation of high molecular weight complexes.

DNA↗
PubMed1999

Probing the structure of multi-stranded guanine-rich DNA complexes by Raman spectroscopy and enzymatic degradation.

The multi-stranded DNA complexes formed by the oligonucleotides d(T15G4T2G4), Tel, and d(T15G15), TG, were examined by nuclease digestion and Raman spectroscopy. Both Tel and TG can aggregate to form structures consisting of multiple, parallel-oriented DNA strands with two independent structural domains. Overall, the structures of the TG and Tel aggregates appear similar. According to the Raman data, the majority of bases are in C2'-endo/anti conformation. The interaction of guanines at the 3'-ends in both complexes stabilizes the complexes and protects them from degradation by exonuclease III. The 5'-extensions remain single-stranded and the thymines are accessible to single-strand-specific nuclease digestion. The extent of enzymatic cleavage at the junction at the 5' end of the 15 thymines implies a conformational change between this part of the molecule and the guanine-rich region. The differential enzymatic sensitivity of the complexes suggests there are variations in backbone conformations between TG and Tel aggregates. TG aggregates were more resistant to digestion by DNase I, Mung Bean nuclease, and S1 nuclease than Tel complexes. It is proposed that the lower DNase I sensitivity may be partly due to the more stable backbone exhibited by TG than Tel complexes. Structural uniformity along the guanine core of TG is suggested, as there is no indication of structural discontinuities or protected sites in the guanine-rich regions of TG aggregates. The lower extent of digestion by Mung Bean nuclease at the 3' end implies that these bases are inaccessible to the enzyme. This suggests that there is minimal fraying at the ends, which is consistent with the extreme thermal stability of the TG aggregates.

Base Composition↗
PubMed1999

High pressure fourier transform infrared spectroscopy of poly(dA)poly(dT), poly(dA) and poly(dT).

The effect of hydrostatic pressure upon the DNA duplex, poly(dA)poly(dT), and its component single strands, poly(dA) and poly(dT) has been studied by fourier-transform infrared spectroscopy (FT-IR). The spectral data indicate that at 28 degrees C and pressures up to 12 kbar (1200 MPa) all three polymers retain the B conformation. Pressure causes the band at 967 cm(-1), arising from water-deoxyribose interactions, to shift to higher frequencies, a result consistent with increased hydration at elevated pressures. A larger pressure-induced frequency shift in this band is observed in the single stranded polymers than in the double stranded molecule, suggesting that the effect of pressure on the hydration of single strands may be greater than upon a double stranded complex. A pressure-dependent hypochromicity in the bands attributed to base stacking indicates that pressure facilitates the base stacking in the three polymers, in agreement with previous assessments of the importance of stacking in the stabilization of DNA secondary structure at ambient and high pressures.

Journal Article↗
PubMed1999

Transient association of the DNA-ligand complex during gel electrophoresis.

DNA frayed wires are extremely stable multistranded complexes arising from the association of oligonucleotides with long terminal runs of consecutive guanines. Frayed wires originating from d(A15G15) have multiple binding sites for short complementary oligonucleotides such as dT10. We examine unusual band patterns obtained when complexes formed between dT10 and DNA frayed wires are resolved on nondenaturing polyacrylamide gels. Since the lifetime of the dT10-frayed wire complexes is shorter than the time of the gel run, the interaction between the components during the gel electrophoresis affects their band patterns. We have conducted chasing experiments to show that (i) the binding of dT10 to the frayed wires can occur during gel electrophoresis, and (ii) dissociation of the complexes occurs during the gel run. Rapid repetitive dissociation-reassociation of the complexes leads to a constant partitioning of dT10 between their binding sites within frayed wires. Consequently, complexes composed of frayed wires and various numbers of bound ligands appear on the gel as a single well-defined band. The mobilities of these bands decrease continuously with the concentration of the ligand reaching saturation when all the binding sites are occupied. This characteristic pattern is observed only for relatively unstable interactions. Longer ligands, i.e., oligonucleotides with higher affinity towards the binding sites, cease to exhibit the dynamic character of interaction during gel electrophoresis. These ligands form long-lived complexes with the frayed wires that appear on the gel as faint smeared bands reflecting the presence of multiple stable complexes.

Binding Sites↗
PubMed1998

Analysis of the electrophoretic migration of DNA frayed wires.

We analyzed the electrophoretic behaviour of the unusual multi-stranded DNA complexes, frayed wires, in polyacrylamide gels under non-denaturing conditions. Frayed wires arise from the association of several strands of a parent oligonucleotide that possesses long terminal runs of consecutive guanines. According to the structural model proposed for frayed wires, there are two distinct conformational domains, a guanine stem and single stranded arms displaced from the stem. The presence of the two domains affects the electrophoretic migration of the frayed wires, resulting in a greater retardation compared to that of double stranded DNA of the same molecular weight. The degree of retardation is determined by the relative length of the stem and the arms; the complexes with longer arms display a stronger dependence on the total molecular weight. Reptation plots (mobility x molecular weight vs. molecular weight) were used to study the electrophoretic behaviour of frayed wires that arise from the different parent oligonucleotides. The plots are unique for each type of frayed wire. The characteristic parameter, the position of the maximum of the reptation plot, depends on the type of the frayed wire as well as the total gel concentration. The plots become similar when we replot the mobility data taking into account only the single stranded arms of the frayed wires. The positions of the maximum and the overall shape are very close for the four types of frayed wires studied.

Chemical Phenomena↗
PubMed1998

Quantitative hydroxyl radical footprinting reveals cooperative interactions between DNA-binding subdomains of PU.1 and IRF4.

Quantitative hydroxyl radical footprinting and fluorescence polarization measurements have been used to determine the dissociation constants (Kd) of complexes between the ets domain of the murine transcription factor PU.1 and three different DNA fragments. Two natural PU.1 binding sites, the SV40 enhancer site and the lambdaB motif of Iglambda2-4 enhancer, were used as well as the PU.1 binding site present in the crystallized PU.1-DNA complex. With the use of quantitative hydroxyl radical footprinting we obtained binding isotherms for individual protected nucleotides and contact sites on both strands of the DNA. Kd values of (1.53 +/- 0. 12) x 10(-)8 M were found for the lambdaB element, (3.60 +/- 0.65) x 10(-)8 M for the SV40 enhancer site, and (2.28 +/- 0.27) x 10(-)8 M for the sequence used in the crystal structure. In addition, the binding of a second protein, the DNA binding domain of IRF4, to the lambdaB site by itself and in the presence of PU.1 was analyzed. The IRF4 DBD shows three footprints on the TTCC strand and one footprint on the GGAA strand of the lambdaB element. The dissociation constant for the binary IRF4 DBD-lambdaB complex equals (5.59 +/- 0.60) x 10(-)7 M. The Kd value of the IRF4-lambdaB interaction is reduced by a factor of 5 in the presence of two different DNA-bound PU.1 protein constructs, PU.1 DBD and a PU.1 construct containing the PEST domain (PU.1-PEST). A similar decrease of the Kd value was observed for the binding of PU.1-PEST in the presence of DNA-bound IRF4 DBD demonstrating a cooperative interaction between the PU. 1-PEST and IRF4 DBD. On the basis of the hydroxyl radical footprints in the ternary PU.1/IRF4/lambdaB complex, a model for the interactions between the two proteins and the lambdaB site was developed. The DNA binding domains of both proteins bind the DNA in the major groove with potential protein-protein interactions near the intervening minor groove.

Animals↗
PubMed1998

Hydroxyl radical footprinting of DNA complexes of the ets domain of PU.1 and its comparison to the crystal structure.

Hydroxyl radical footprinting has been used to probe interactions in complexes between the ets domain of the murine transcription factor PU.1 and three different DNA restriction fragments, each containing one copy of the recognition sequence 5'-GGAA-3'. Two natural PU.1 binding sites, the SV40 enhancer site and the lambdaB motif of Ig lambda2-4 enhancer, were used as well as the PU.1 binding site present in the crystallized PU.1-DNA complex [Kodandapani, R., Pio, F., Ni, C.-Z., Piccialli, G., Klemsz, M., McKercher, S. R., Maki, R. A., and Ely, K. R. (1996) Nature 380, 456-460]. The footprints obtained for the three different DNA sequences are almost identical. The extent of contact with the protein was monitored for every base in the complex. Two concentration-dependent cleavage sites on the complementary TTCC strand are evidence of a specific interaction between PU.1 and the DNA. Two more protection sites and a hypersensitive cleavage site on the GGAA strand were observed. Although these data confirm the global structure of the PU.1-DNA complex as suggested by crystallography, the footprinting data reveal differences between the protein-DNA contacts in solution and in the crystal state. An additional interaction site not present in the crystal structure was observed by hydroxyl radical footprinting.

Animals↗
PubMed1998

Effect of hydrostatic pressure on nucleic acids.

In comparison to other biomolecules, the effect of hydrostatic pressure on the conformational stability of DNA and RNA has received scant attention. However, the increasing interest in the hydration of biological molecules has resulted in a concomitant increase in the number of investigations of the effect of pressure upon the structure of nucleic acids. In this review, studies concerning the effect of pressure on DNA and RNA oligomers and polymers are presented. The greatest amount of research has been directed at studying the effect of pressure on the stability of the double helix. In general, under most conditions, the helical form of DNA or RNA is stabilized by pressure. The extent of stabilization is small relative to the effect of pressure on other biomolecular systems such as lipid membranes or protein quaternary structure. The absence of a larger pressure effect arises, in part because the state of ionization does not change as a function of the helical state. Initial experiments have also appeared on the effect of pressure upon helix-formation kinetics, B-Z and A-Z equilibria, and DNA topology. Fourier-transform ir spectroscopy of DNA polymers under high pressure has yielded data that showing that pressure does not induce large-scale structural changes.

Animals↗
PubMed1998

Unusual behavior exhibited by multistranded guanine-rich DNA complexes.

The structural properties of oligonucleotides containing two different types of G-rich sequences at the 3'-ends were compared. It is shown that oligonucleotides with uninterrupted runs of guanine residues at the 3'-end, e.g., d(T15G12), form multistranded structures stabilized by guanine-guanine interactions. The chemical and physical properties of these complexes differ from those of the complexes formed by oligonucleotides with telomere-like sequences, e.g., d(T15G4T2G4). In methylation protection and methylation interference experiments, we found all the guanines in complexes formed by d(T15G15) and d(T15G12) to be accessible to methylation. Furthermore, the methylated monomers retain the ability to polymerize. This contrasts with the inaccessibility of the guanines in d(T15G4T2G4) to methylation and the inability of the methylated monomer to form supramolecular structures. The stoichiometry of the complexes arising from the two types of oligonucleotides also differs. The complexes formed by d(T15G15) consist of consecutive integer numbers of DNA strands, whereas complexes formed by telomere-like oligonucleotides contain 1, 2, 4, or multiples of four strands. Magnesium ions favor formation of high molecular weight complexes by d(T15G15) and d(T15G12), but not by d(T15G4T2G4). The d(T15G15) and d(T15G12) complexes have very high thermal stability compared with telomeric complexes. However, at low temperatures, the thymine bases within the telomeric motif, TTGGGGTTGGGG, appear to allow for the formation of stable high-molecular weight species with a longer nonguanine portion.

DNA↗
PubMed1998

Circular dichroism of DNA frayed wires.

Ultraviolet circular dichroism spectra are reported for the oligonucleotide d(A15G15) in aqueous solutions containing 5 mM MgCl2 at several temperatures and in the presence of partially complementary oligonucleotides. Oligonucleotides with several consecutive terminal guanine residues self-associate to form aggregates, called frayed wires, that consist of integer numbers of strands. A "stem" is formed through interactions between the guanine residues of the associated oligonucleotides, whereas the adenine "arms" remain single stranded. Upon subtracting the circular dichroism spectrum of d(A15) from that of d(A15G15), one obtains a spectrum that closely resembles previously published spectra of poly(G). Subtracting spectra measured at temperatures between 10 degrees C and 60 degrees C reveals the resultant spectra to be independent of temperature, consistent with the extreme thermal stability observed for the aggregated structures. Upon the addition of d(T15) to the solution, complexes with the adenine portion of the d(A15G15) frayed wires are formed. Subtraction of d(A15):d(T15) spectra measured at several temperatures from those of the d(A15G15):d(T15) does not significantly alter the spectrum of the guanines. The helix-coil transition temperature of d(A15):d(T15) duplex is identical to that of the unbinding of d(T15) from d(A15G15):d(T15) complexes. Experiments using oligonucleotides in which the adenines were replaced with sequences of bases yielded similar results. By varying the length of the nonguanine tract, it is shown that the solubility of the complexes increases with the length of the nonguanine region of the oligonucleotide.

Adenine↗
PubMed1997

Activation volume of DNA duplex formation.

The denaturation-renaturation thermal hysteresis was used to investigate the kinetics of the helix-coil equilibrium of four 22-base pair homopurine-homopyrimidine duplex oligonucleotides with fractional G x C base pair content (f(G x C)) between 0.14 and 0.5. In 20 mM NaCl and 20 mM Tris-HCl at pH 7.0 and at hydrostatic pressures up to 200 MPa, a two-state bimolecular reaction mechanism adequately described the observed kinetics. At 1 MPa and 47 degrees C, the rate constant for helix formation, k1, increased by a factor of 210, and the reverse rate constant, k(-1), decreased by a factor of 420 upon increasing f(G x C) from 0.14 to 0.5. The activation energies for formation of the duplexes were negative and relatively insensitive to f(G x C). The pressure-induced change in the rate constants is related to the activation volume of the reaction step. Pressure causes k1 to become larger, and the magnitude of the change in k1 with pressure increases the lower the f(G x C) value. Thus, when f(G x C) = 0.14, the activation volume for forward reaction, delta V++(1), equals -20 mL/mol, while when f(G x C) = 0.5, delta V++(1) = -6.7 mL/mol. The rate constant for strand separation, k(-1), decreases at high pressure. The activation volume for this step, delta V++(1), varies from 17 to 1.6 mL/mol when f(G x C) = 0.14 and 0.5, respectively. The delta V for helix formation calculated from the activation parameters changed from -23 mL/mol when f(G x C) = 0.14 to -5.8 mL/mol when f(G x C) = 0.5. From extrapolation, it is estimated that the molar volume change for formation of G x C base pairs in homopurine-homopyrimidine sequences is approximately 0 mL/mol. Parameters calculated from kinetics of other two duplex molecules, when f(G x C) = 0.23 and 0.32, lie between these extremes.

Base Composition↗
PubMed1996

Frayed wires: a thermally stable form of DNA with two distinct structural domains.

In aqueous solutions containing mono- and divalent cations, the oligodeoxyribonucleotide d(A15G15) readily self-assembles into high-molecular weight species that resolve as discrete bands on native and denaturing electrophoresis gels. The complexes consist of an integer number of strands of d(A15G15), the number of strands range from one (the monomer) to greater than nine. The complexes form within a few minutes even at low concentrations (2 microM strands) of d(A15G15). The relative concentration of species is determined by the solvent conditions. The complexes are resistant to standard denaturation conditions, 50% formamide heated to 95 degrees C for 2 min followed by electrophoresis in 7 M urea at 55 degrees C. In the proposed model for the oligomers and polymers of d(A15G15), several molecules of d(A15G15) interact via a stem of tetraplex structure formed by the guanine residues. The 15 guanine residues in the stem account for its high stability. The 5' end adenines form single-stranded arms that are displaced from the guanine-containing stem. The arms can participate in the formation of Watson-Crick base pairs with dT10 and other partially complementary oligodeoxyribonucleotides such as d(CT15). Engagement of the arms in interactions with other strands does not affect the distribution of the species between different conformations. On the other hand, the addition of the fully complementary oligodeoxyribonucleotide d(C15T15) to the polymer leads to the disappearance of the high-molecular weight complexes and results in the formation of a canonical Watson-Crick base-paired duplex. The type and concentration of the cation present in solution determine which conformation d(A15G15) will adopt. Divalent cations at millimolar concentrations lead to the formation of the polymer, while the presence of the monovalent cations stabilizes lower-molecular weight complexes consisting of two to six strands of d(A15G15).

Base Sequence↗
PubMed1996

Kinetic footprinting of DNA triplex formation.

Kinetic parameters of triplex-forming reaction between 22-base-pair duplex oligonucleotide (5'-d[AAAGGAGGAGAAGAAGAAAAAA], sequence of purine strand) and the third strand 5'-d[TTTCCTCCTCTTCTTCTTTTTT] were determined by quantitative footprinting using DNase I as the cleavage reagent. When the third strand oligonucleotide is present in 10-fold excess over its duplex target, the binding reaction kinetics is pseudo first order in oligonucleotide concentration. Under the conditions of these measurements (10 mM sodium cacodylate, pH 6.9, 2 mM MgCl2), the reactions are slow with relaxation times on the order of minutes (4 to 28 min). As is generally found for helix-formation reactions, the forward rate constant (helix formation) decreased with temperature, the bimolecular association rate constants ranged from 237 M-1 s-1 at 10 degrees C to 13 M-1 s-1 at 30 degrees C. These data are consistent with an activation energy of -25 kcal/mol (strands). The dissociation rate constant apparently is temperature independent under these conditions; the changes observed were within the error that this parameter could be determined. Advantages and limitations of this technique for obtaining the kinetic parameters of reactions involving sequence-specific DNA complexes are discussed. The technique can be readily implemented in most biochemistry or molecular biology laboratories.

DNA↗
PubMed1996

The activation volume of a DNA helix-coil transition.

The role of hydration in the kinetics of a DNA helix-coil equilibrium is investigated by studying the effect of hydrostatic pressure on the rate constants describing the reaction. The kinetics were measured using the thermal hysteresis between the denaturation and renaturation curves of the triplex-forming oligonucleotides: 5'd[AAA-GGAGGAGAAGAAGAAAAAA] (sequence of purine strand) and 5'd[TTTCCTCCTCTTCTTCTTTTTT] (third strand). The kinetics at atmosphere pressure for this system have been recently reported [Rougée et al. (1992) Biochemistry 31, 9269-9278]. At all pressures the data are consistent with a single-step bimolecular reaction under the conditions of our experiments (100 mM NaCl, 10 mM cacodylate, pH 6.5). The rate of formation of the triplex from the duplex + single strand is accelerated by pressure. At the midpoint of the helix-coil transition (32.5 degrees C), the activation volume for helix formation, V*1, equals -11.8 (+/- 2.4) cm3 mol-1 at atmospheric pressure. At the same temperature, the activation volume for helix dissociation, V*-1, equals +39.9 (+/- 5.0) cm3 mol-1; that is, the rate of strand separation is slowed by pressure. These findings emphasize the importance of solvent interactions in the stabilization and formation of DNA helices. It is proposed that the activation volume of the forward reaction may arise from the volume change due to charging the cytosine residues and the formation of base-stacking interactions in the third strand. The positive activation volume of strand separation may be a consequence of poor solvent packing of the DNA duplex major groove during dissociation of the third strand.

Base Sequence↗