Papers by Nancy Stellwagen
Biochemistry, Jun 6, 2013
The intrinsic curvature of seven 98-base pair DNA molecules containing up to four centrally locat... more The intrinsic curvature of seven 98-base pair DNA molecules containing up to four centrally located A 6 -tracts has been measured by gel and capillary electrophoresis as a function of the number and arrangement of the A-tracts. At low cation concentrations, the electrophoretic mobility observed in polyacrylamide gels and in free solution decreases progressively with the increasing number of phased A-tracts, as expected for DNA molecules with increasingly curved backbone structures. Anomalously slow electrophoretic mobilities are also observed for DNA molecules containing two pairs of phased A-tracts that are out of phase with each other, suggesting that out-of-phase distortions of the helix backbone do not cancel each other out. The mobility decreases observed for the A-tract samples are due to curvature, not cation binding in the A-tract minor groove, because identical free solution mobilities are observed for a molecule with four out-of-phase A-tracts and one with no A-tracts. Surprisingly, the curvature of DNA A-tracts is gradually lost when the monovalent cation concentration is increased to ~200 mM, regardless of whether the cation is a hydrophilic ion like Na + , NH 4 + or Tris + or a hydrophobic ion like tetrabutylammonium (TBA + ). The decrease of A-tract curvature with increasing ionic strength, along with the known decrease of A-tract curvature with increasing temperature, suggests that DNA A-tracts are not significantly curved under physiological conditions.
Monovalent cation localization in DNA A‐tracts with different sequences
Electrophoresis, Jun 24, 2023
The free solution mobilities of 26‐base pair (bp) DNA oligomers containing A‐tracts with and with... more The free solution mobilities of 26‐base pair (bp) DNA oligomers containing A‐tracts with and without internal ApT steps have been measured by capillary electrophoresis, using the mobility of a 26‐bp random‐sequence oligomer as a reference. The background electrolytes (BGEs) contained mixtures of Li+ and tetrapropylammonium (TPA+) ions, keeping the total cation concentration constant at 0.3 M. The mobility ratios equaled 1.00 in 0.3 M TPA+, indicating that the A‐tract and reference oligomers had the same B‐form conformation in this BGE. With increasing [Li+], the mobility ratio decreased as Li+ ions became localized in the A‐tract minor groove, suggesting that the A‐tract was now in the B* conformation. If the A‐tract contained an internal ApT step and the oligomer contained less than ∼50% A + T, the mobility ratio reached a reduced plateau value that remained constant as the [Li+] increased to 0.3 M. However, for A‐tracts without an internal ApT step and for A‐tracts embedded in oligomers containing more than 50% A + T, the mobility ratios increased again at high [Li+], eventually reaching a plateau value of 1.00. Hence, DNA A‐tracts in solution appear to exist as mixtures of the B and B* conformations, with the fractional concentration of each conformer depending on the [Li+], the A‐tract sequence, and the total A + T content of the oligomer.
Biophysical Journal, Mar 1, 2008
Monovalent cation binding by DNA A-tracts, runs of four or more contiguous adenine or thymine res... more Monovalent cation binding by DNA A-tracts, runs of four or more contiguous adenine or thymine residues, has been determined for two curved ;200 basepair (bp) restriction fragments, one taken from the M13 origin of replication and the other from the VP1 gene of SV40. These two fragments have previously been shown to contain stable, centrally located bends of 44°and 46°, respectively, located within ;60 bp ''curvature modules'' containing four or five irregularly spaced A-tracts. Transient electric birefringence measurements of these two fragments, sequence variants containing reduced numbers of A-tracts in the SV40 curvature module or changes in the residues flanking the A-tracts in the M13 curvature module, have been combined with the free solution electrophoretic mobilities of the same fragments using known equations to estimate the effective charge of each fragment. The effective charge is reduced, on average, by one-third charge for each A-tract in the curvature module, suggesting that each A-tract binds a monovalent cation approximately one-third of the time. Monovalent cation binding to two or more A-tracts is required to observe significant curvature of the DNA helix axis.
Journal of Molecular Biology, Feb 1, 2001
The free solution mobility of four 20 bp DNA oligomers, with and without A-tracts, has been measu... more The free solution mobility of four 20 bp DNA oligomers, with and without A-tracts, has been measured by capillary electrophoresis in Trisacetate buffer, to test the hypothesis that site-speci®c binding of monovalent counterions can occur in the narrow minor groove of A-tract DNAs. Preferential counterion binding has been proposed to cause A-tract bending because of asymmetric charge neutralization and collapse of the helix backbone toward the minor groove. Preferential counterion binding in A-tract DNAs should be manifested by a decrease in the electrophoretic mobility observed in free solution, compared to that of non-A-tract DNAs of the same size. Of the four sequences studied here, the slowest absolute mobility, indicative of the greatest counterion binding, was observed for a 20 bp oligomer containing two runs of A 3 T 3 in phase with the helix repeat. A 20-mer containing phased CACA sequences migrated with the fastest mobility; 20-mers containing phased A 5 tracts or phased runs of T 3 A 3 migrated with intermediate mobilities. Very similar mobility differences were observed when 1-20 mM NaCl was added to the buffer. The results suggest that preferential counterion binding occurs in A-tract DNAs, especially those containing the A n T n sequence motif.
Biophysical Journal, 2014
Biophysical Journal, May 1, 2015
Capillary electrophoresis has been used to characterize the interaction of monovalent cations wit... more Capillary electrophoresis has been used to characterize the interaction of monovalent cations with 26-basepair DNA oligomers containing A-tracts embedded in flanking sequences with different basepair compositions. A 26-basepair random-sequence oligomer was used as the reference; lithium and tetrabutylammonium (TBA þ ) ions were used as the probe ions. The free solution mobilities of the A-tract and random-sequence oligomers were identical in solutions containing <~100 mM cation. At higher cation concentrations, the A-tract oligomers migrated faster than the reference oligomer in TBA þ and slower than the reference in Li þ . Hence, cations of different sizes can interact very differently with DNA A-tracts. The increased mobilities observed in TBA þ suggest that the large hydrophobic TBA þ ions are preferentially excluded from the vicinity of the A-tract minor groove, increasing the effective net charge of the A-tract oligomers and increasing the mobility. By contrast, Li þ ions decrease the mobility of A-tract oligomers because of the preferential localization of Li þ ions in the narrow A-tract minor groove. Embedding the A-tracts in AT-rich flanking sequences markedly alters preferential interactions of monovalent cations with the B* conformation. Hence, A-tracts embedded in genomic DNA may or may not interact preferentially with monovalent cations, depending on the relative number of A$T basepairs in the flanking sequences.
Biochemistry, Jan 20, 2009
The binding of five different monovalent cations to DNA oligomers containing A-tracts, runs of fo... more The binding of five different monovalent cations to DNA oligomers containing A-tracts, runs of four or more contiguous adenine residues, has been measured by capillary electrophoresis, using the Replacement Ion method. In this method, a non-binding cation in the background electrolyte is gradually replaced by a binding cation, keeping the ionic strength of the solution constant. Monovalent cation binding reduces the effective charge of an A-tract-containing soligomer, decreasing its free solution mobility. The cations bind in the A-tract minor groove, because the binding site can be blocked by the minor groove binding drug netropsin. Li + , NH 4 + and Tris + ions bind to A-tracts with similar affinities; the binding of Na + ions is weaker, and K + ion binding is highly variable. Each A-tract appears to bind one monovalent cation upon saturation of the binding site(s). For a given cation, the apparent dissociation constants depend on A-tract sequence and orientation, but not on the phasing of the A-tracts with respect to the helix repeat. Differences in the cooperativity of binding of the various cations to A-tracts with different sequences suggest that monovalent cation binding may be coupled with a conformational transition leading to the formation of the characteristic narrow minor groove A-tract structure.
Circular dichroism and thermal melting of two small DNA restriction fragments of the same molecular weight
Biochemistry, Dec 18, 1984
The thermal melting and circular dichroism of two 147 base pair restriction fragments of pBR322 h... more The thermal melting and circular dichroism of two 147 base pair restriction fragments of pBR322 have been studied. The fragment with the higher GC content, 12B, melts at a higher temperature than the other fragment, 12A, as expected. The melting temperatures are proportional to the logarithm of the concentration of NaCl or tris(hydroxymethyl)aminomethane (Tris) buffer, between 1 mM and 0.2 M added salt. In 1 mM Tris buffer, the melting temperatures of the two fragments are nearly equal. The circular dichroism spectra of fragments 12A and 12B in 0.2-10 mM Tris buffer are characteristic of B-form DNA. In 81% ethanol, the circular dichroism spectra of the two fragments are characteristic of A-form DNA. With 1 mM Tris buffer as the supporting electrolyte, fragment 12A exhibits a very sharp B----A transition, with a midpoint at 79% ethanol. However, a biphasic transition is observed for fragment 12B, with midpoints at 73% and 80% ethanol. This biphasic transition may represent the conversion of separate domains of fragment 12B from the B conformation to the A conformation; half of this fragment is much more GC rich than the other half. Methods are also described for preparing polymers of the 12A and 12B fragments.
Biophysical Journal, 2014
Biophysical Journal, 2015
Capillary electrophoresis has been used to characterize the interaction of monovalent cations wit... more Capillary electrophoresis has been used to characterize the interaction of monovalent cations with 26-basepair DNA oligomers containing A-tracts embedded in flanking sequences with different basepair compositions. A 26-basepair random-sequence oligomer was used as the reference; lithium and tetrabutylammonium (TBA þ ) ions were used as the probe ions. The free solution mobilities of the A-tract and random-sequence oligomers were identical in solutions containing <~100 mM cation. At higher cation concentrations, the A-tract oligomers migrated faster than the reference oligomer in TBA þ and slower than the reference in Li þ . Hence, cations of different sizes can interact very differently with DNA A-tracts. The increased mobilities observed in TBA þ suggest that the large hydrophobic TBA þ ions are preferentially excluded from the vicinity of the A-tract minor groove, increasing the effective net charge of the A-tract oligomers and increasing the mobility. By contrast, Li þ ions decrease the mobility of A-tract oligomers because of the preferential localization of Li þ ions in the narrow A-tract minor groove. Embedding the A-tracts in AT-rich flanking sequences markedly alters preferential interactions of monovalent cations with the B* conformation. Hence, A-tracts embedded in genomic DNA may or may not interact preferentially with monovalent cations, depending on the relative number of A$T basepairs in the flanking sequences.
ACS Symposium Series, 2004
The structure of DNA proposed by Watson and Crick (1) from fiber diffraction data (2) was symmetr... more The structure of DNA proposed by Watson and Crick (1) from fiber diffraction data (2) was symmetric and highly regular, leading to the idea that DNA structure was monolithic and independent of sequence. Subsequent studies, however, especially by Arnott and coworkers (3), showed that DNA structure is in fact highly variable, and depends on hydration, base-pair composition and the identity of the counterion. Many of the early investigations of DNA conformation in solution revolved around the questions of polymer physics-the size, shape, and flexibility of the macromolecule and its stability under a variety of experimental conditions (reviews: 4-7). The modern era of DNA structural studies began when the x-ray crystal structure of a dodecamer containing the EcoRI recognition site was solved by . This structure showed that each base-pair step makes its own individual contribution to the conformation of the helix, indicating that DNA structure must be sequencedependent. A host of other x-ray and NMR studies of small DNA oligomers and DNA-drug and DNA-protein complexes followed, providing atomic-level detail about DNA structure and conformation in a variety of contexts (reviews: 6, 7, 11-14). These and other studies were made possible by concomitant advances in molecular biology and the development of methods for automated oligonucleotide synthesis, which made it possible to prepare the large quantities of highly purified DNA oligomers needed for biophysical studies.
Journal of Molecular Biology, 2001
The free solution mobility of four 20 bp DNA oligomers, with and without A-tracts, has been measu... more The free solution mobility of four 20 bp DNA oligomers, with and without A-tracts, has been measured by capillary electrophoresis in Trisacetate buffer, to test the hypothesis that site-speci®c binding of monovalent counterions can occur in the narrow minor groove of A-tract DNAs. Preferential counterion binding has been proposed to cause A-tract bending because of asymmetric charge neutralization and collapse of the helix backbone toward the minor groove. Preferential counterion binding in A-tract DNAs should be manifested by a decrease in the electrophoretic mobility observed in free solution, compared to that of non-A-tract DNAs of the same size. Of the four sequences studied here, the slowest absolute mobility, indicative of the greatest counterion binding, was observed for a 20 bp oligomer containing two runs of A 3 T 3 in phase with the helix repeat. A 20-mer containing phased CACA sequences migrated with the fastest mobility; 20-mers containing phased A 5 tracts or phased runs of T 3 A 3 migrated with intermediate mobilities. Very similar mobility differences were observed when 1-20 mM NaCl was added to the buffer. The results suggest that preferential counterion binding occurs in A-tract DNAs, especially those containing the A n T n sequence motif.
ELECTROPHORESIS, 2014
The free solution mobilities of ssDNA and dsDNA molecules with variable charge densities have bee... more The free solution mobilities of ssDNA and dsDNA molecules with variable charge densities have been measured by CE. DNA charge density was modified either by appending positively or negatively charged groups to the thymine residues in a 98 bp DNA molecule, or by replacing some of the negatively charged phosphate internucleoside linkers in small ssDNA or dsDNA oligomers with positively charged phosphoramidate linkers. Mobility ratios were calculated for each dataset by dividing the mobility of a charge variant by the mobility of its unmodified parent DNA. Mobility ratios essentially eliminate the effect of the BGE on the observed mobility, making it possible to compare analytes measured under different experimental conditions. Neutral moieties attached to the thymine residues in the 98‐bp DNA molecule had little or no effect on the mobility ratios, indicating that bulky substituents in the DNA major groove do not affect the mobility significantly. The mobility ratios observed for the ...
Quantitative analysis of cation binding to the adenosine nucleotides using the Variable Ionic Strength method: Validation of the Debye–Hückel–Onsager theory of electrophoresis in the absence of counterion binding
ELECTROPHORESIS, 2007
The free solution mobilities of the adenosine nucleotides 5&amp;amp;amp;amp;amp;amp;#39;-aden... more The free solution mobilities of the adenosine nucleotides 5&amp;amp;amp;amp;amp;amp;#39;-adenosine triphosphate (ATP), 5&amp;amp;amp;amp;amp;amp;#39;-adenosine diphosphate (ADP), 5&amp;amp;amp;amp;amp;amp;#39;-adenosine monophosphate (AMP), and 3&amp;amp;amp;amp;amp;amp;#39;-5&amp;amp;amp;amp;amp;amp;#39;-cyclic AMP (cAMP) have been measured in diethylmalonate buffers containing a wide variety of monovalent cations. The mobilities of all nucleotides increase gradually with the increase in intrinsic conductivity of the cation in the BGE. However, at a given conductivity, the mobilities observed for ATP, ADP, and AMP in BGEs containing alkali metal ions and other cations are lower than these observed in BGEs containing tetraalkylammonium ions. Since the mobility of cAMP is independent of the cation in the BGE, the results suggest that the relatively low mobilities observed for ATP, ADP, and AMP in BGEs containing cations other than a tetraalkylammonium ion are due to cation binding, reducing the effective net charge of the nucleotide and thereby reducing the observed mobility. To measure the binding quantitatively, the mobilities of the nucleotides were measured as a function of ionic strength. The mobilities of ATP, ADP, and AMP decrease nonlinearly with the square root of ionic strength (I(1/2)) in BGEs containing an alkali metal ion or Tris(+). By contrast, the mobilities decrease linearly with I(1/2) in BGEs containing a nonbinding quaternary ammonium ion, as expected from Debye-Hückel-Onsager (DHO) theory. The mobility of cAMP, a nonbinding analyte, decreases linearly with I(1/2), regardless of the cation in the BGE. Hence, a nonlinear decrease of the mobility of an analyte with I(1/2) appears to be a hallmark of counterion binding. The curved mobility profiles observed for ATP, ADP, and AMP in BGEs containing an alkali metal ion or Tris(+) were analyzed by nonlinear curve fitting, using difference mobility profiles to correct for the effect of the physical properties of BGE on the observed mobilities. The calculated apparent dissociation constants range from 22 to 344 mM, depending on the particular cation-nucleotide pair. Similar values have been obtained by other investigators, using different methods. Interestingly, Tris(+) and Li(+) bind to the adenosine nucleotides with approximately equal affinities, suggesting that positively charged Tris(+) buffer ions can compete with alkali metal ions in Tris-buffered solutions.
ELECTROPHORESIS, 1998
When separating ds‐DNA in isoelectric His buffer (pHpI7.6), in the 50–250 mM concentration rang... more When separating ds‐DNA in isoelectric His buffer (pHpI7.6), in the 50–250 mM concentration range, some unique phenomena were observed: improved resolution for smaller DNA fragments, up to ca. 150 bp, and a rapid deterioration of resolution above this critical length (which corresponds to the persistence length). Such phenomenon depended also on voltage and concentration of sieving liquid polymer. Direct binding of His to the DNA helix was hypothesized, with resultant stiffening and an increment of diameter of the DNA fragments, thus inducing an early onset of reptation at the applied voltage in the 100–300 V/cm range. In order to prove this hypothesis, “competing ions” (notably NaCl and KBr) were added to the His background electrolyte: a partial reversal of the His effect was already apparent at low concentrations of such ions (10 mM) and was complete at higher concentrations (30 and 50 mM). By molecular modeling, it was found that His could be docking on the negatively charged o...
Effect of organic cosolvents on the free solution mobility of curved and normal DNA molecules
ELECTROPHORESIS, 2006
The free solution mobilities of curved and normal 199‐bp DNA fragments have been measured in buff... more The free solution mobilities of curved and normal 199‐bp DNA fragments have been measured in buffer solutions containing various quantities of the organic cosolvents methanol, ethanol, 2‐propanol, 2‐methyl‐2,4‐pentanediol (MPD), ethylene glycol, and ACN, using CE. The curved fragment, taken from the VP1 gene of SV40, contains five unevenly spaced A‐ and T‐tracts in a centrally located “curvature module”; the A‐ and T‐tracts have been mutated to other sequences in the normal 199‐bp fragment. The free solution mobility of the curved 199‐bp fragment is significantly lower than that of its normal counterpart in aqueous solutions [Stellwagen, E., Lu, Y. J., Stellwagen, N. C., Nucleic Acids Res. 2005, 33, 4425–4432]. The mobilities of both the curved and normal fragments decrease with increasing cosolvent concentration, due to the effect of the cosolvent on the viscosity and dielectric constant of the solution. The mobility differences between the curved and normal 199‐bp fragments and th...
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Papers by Nancy Stellwagen