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Nancy C Stellwagen

Publications and source records attributed to Nancy C Stellwagen.

15 recordsLinked to original sources
PubMed2006

Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration.

The electrophoretic mobilities of curved and normal DNA molecules of the same size have been measured in polyacrylamide gels containing various acrylamide concentrations and cross-linker ratios. Ferguson plots were constructed to extrapolate the observed mobilities to zero gel concentration. The DNA samples were two 147-bp restriction fragments, called 12A and 12B, obtained from the MspI digestion of plasmid pBR322, and head-to-tail multimers of each fragment. Fragment 12A is stably curved and migrates anomalously slowly in polyacrylamide gels; fragment 12B has the conformation of normal DNA and migrates with normal electrophoretic mobilities. The extrapolated mobilities of the curved fragment 12A and its multimers at zero gel concentration are lower than the extrapolated mobilities of the normal fragment 12B and its multimers. The free solution mobility of the curved fragment 12A, measured by CE, is also lower than that of the normal fragment 12B. The combined results indicate that the extrapolated mobilities observed for curved DNA molecules at zero polyacrylamide gel concentration reflect the intrinsic differences in their free solution mobilities.

Base Sequence↗
PubMed2006

Effect of organic cosolvents on the free solution mobility of curved and normal DNA molecules.

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 the mobility ratios decrease approximately linearly with the increasing mole fraction of cosolvent in the solution. Hence, MPD and other organic cosolvents affect DNA electrophoretic mobility by a common mechanism, most likely the preferential hydration of the DNA surface that occurs in aqueous cosolvents. The gradual loss of the anomalously slow mobility of the curved 199-bp fragment with increasing cosolvent concentration, combined with other data in the literature, suggests that preferential hydration gradually widens the narrow A-tract minor groove, releasing site-bound counterions in the minor groove and shifting the conformation toward that of normal DNA.

Alcohols↗
PubMed2005

Curved DNA molecules migrate anomalously slowly in free solution.

The electrophoretic mobility of a curved DNA restriction fragment taken from the VP1 gene in the SV40 minichromosome has been measured in polyacrylamide gels and free solution, using capillary electrophoresis. The 199 bp restriction fragment has an apparent bend angle of 46 +/- 2 degrees located at SV40 sequence position 1922 +/- 2 bp [Lu Y.J., Weers B.D. and Stellwagen N. C. (2005) Biophys. J., 88, 1191-1206]. The 'curvature module' surrounding the apparent bend center contains five unevenly spaced A- and T-tracts, which are responsible for the observed curvature. The parent 199 bp fragment and sequence mutants containing at least one A-tract in the curvature module migrate anomalously slowly in free solution, as well as in polyacrylamide gels. Hence, the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are due in part to their anomalously slow mobilities in free solution. Analysis of the gel and free solution mobility decrements indicates that each A- or T-tract contributes independently, but not equally, to the curvature of the 199 bp fragment and its A-tract mutants. The relative contribution of each A- or T-tract to the observed curvature depends on its spacing with respect to the first A-tract in the curvature module.

AT Rich Sequence↗
PubMed2005

Monovalent cations affect the free solution mobility of DNA by perturbing the hydrogen-bonded structure of water.

The free solution mobilities of single- and double-stranded DNA molecules of various molecular weights have been measured by capillary electrophoresis in solutions of constant ionic strength containing a common anion and fifteen different monovalent cations. In solutions with the same ionic composition, the mobilities of different DNA molecules can vary by up to 20%, depending on molecular weight, the number of strands, and the presence or absence of A-tracts, runs of four or more contiguous adenine residues. Importantly, the mobilities observed for the same DNA sample can vary by up to 40% in solutions containing different cations. The mobility differences observed for the same DNA in solutions containing different cations cannot be rationalized by differences in the anhydrous radii or intrinsic conductivities of the various cations, or by the sequence-dependent binding of certain cations to A-tracts. Instead, the observed mobilities are linearly correlated with the average number of water-water hydrogen bonds that are present in solutions containing different cations. The mobilities are also correlated with the viscosity B coefficients of the various cations and with the rotational correlation times frictional coefficients observed for water molecules in solutions containing different cations. Hence, monovalent cations modify the free solution mobility of DNA primarily by perturbing the hydrogen-bonded structure of water, affecting the friction experienced by the migrating DNA molecules during electrophoresis.

Base Sequence↗
PubMed2004

Intrinsic curvature in the VP1 gene of SV40: comparison of solution and gel results.

DNA restriction fragments that are stably curved are usually identified by polyacrylamide gel electrophoresis because curved fragments migrate more slowly than normal fragments containing the same number of basepairs. In free solution, curved DNA molecules can be identified by transient electric birefringence (TEB) because they exhibit rotational relaxation times that are faster than those of normal fragments of the same size. In this article, the results observed in free solution and in polyacrylamide gels are compared for a highly curved 199-basepair (bp) restriction fragment taken from the VP1 gene in Simian Virus 40 (SV40) and various sequence mutants and insertion derivatives. The TEB method of overlapping fragments was used to show that the 199-bp fragment has an apparent bend angle of 46 +/- 2 degrees centered at sequence position 1922 +/- 2 bp. Four unphased A- and T-tracts and a mixed A3T4-tract occur within a span of approximately 60 bp surrounding the apparent bend center; for brevity, this 60-bp sequence element is called a curvature module. Modifying any of the A- or T-tracts in the curvature module by site-directed mutagenesis decreases the curvature of the fragment; replacing all five A- and T-tracts by random-sequence DNA causes the 199-bp mutant to adopt a normal conformation, with normal electrophoretic mobilities and birefringence relaxation times. Hence, stable curvature in this region of the VP1 gene is due to the five unphased A- and T- tracts surrounding the apparent bend center. Discordant solution and gel results are observed when long inverted repeats are inserted within the curvature module. These insertion derivatives migrate anomalously slowly in polyacrylamide gels but have normal, highly flexible conformations in free solution. Discordant solution and gel results are not observed if the insert does not contain a long inverted repeat or if the long inverted repeat is added to the 199-bp fragment outside the curvature module. The results suggest that long inverted repeats can form hairpins or cruciforms when they are located within a region of the helix backbone that is intrinsically curved, leading to large mobility anomalies in polyacrylamide gels. Hairpin/cruciform formation is not observed in free solution, presumably because of rapid conformational exchange. Hence, DNA restriction fragments that migrate anomalously slowly in polyacrylamide gels are not necessarily stably curved in free solution.

Birefringence↗
PubMed2003

Unified description of electrophoresis and diffusion for DNA and other polyions.

The electrophoretic mobilities and diffusion coefficients of single- and double-stranded DNA molecules up to 50,000 bases or base pairs in size have been analyzed, using mobilities and diffusion coefficients either measured by capillary electrophoresis or taken from the literature. The Einstein equation suggests that the electrophoretic mobilities (mu) and diffusion coefficients (D) should be related by the expression mu/D = Q/k(B)T, where Q is the charge of the polyion (Q = ze(o), where z is the number of charged residues and e(o) is the fundamental electronic charge), k(B) is Boltzmann's constant, and T is the absolute temperature. If this equation were true, the ratio mu/zD should be a constant equal to e(o)/k(B)T (39.6 V(-1)) at 20 degrees C. However, the ratio mu/zD decreases with an increase in molecular weight for both single- and double-stranded DNAs. The mobilities and diffusion coefficients are better described by the modified Einstein equation mu/N(m)D = e(o)/k(B)T, where N is the number of repeat units (bases or base pairs) in the DNA and m is a constant equal to the power law dependence of the diffusion coefficients on molecular weight. The average value of the ratio mu/N(m)D is 40 +/- 4 V(-1) for 36 single- and double-stranded DNA molecules of different sizes, close to the theoretically expected value. The generality of the modified Einstein equation is demonstrated by analyzing literature values for sodium polystyrenesulfonate (PSS). The average value of the ratio mu/N(m)D is 35 +/- 6 V(-1) for 14 PSS samples containing up to 855 monomers.

Base Composition↗
PubMed2003

Analysis of DNA bending by transient electric birefringence.

Transient electric birefringence has been used to analyze DNA bending in six restriction fragments containing 171, 174, 207, 263, 289, and 471 bp in three different low ionic strength buffers. The target fragments contain sequences corresponding to the apparent bend centers in pUC19 and Litmus 28, previously identified by the circular permutation assay (Strutz, K.; Stellwagen, N. C. Electrophoresis 1996, 17, 989-995). The target fragments migrate anomalously slowly in polyacrylamide gels and exhibit birefringence relaxation times that are shorter than those of restriction fragments of the same size, taken from nonbent regions of the same plasmids. Apparent bend angles ranging from 30 degrees to 41 degrees were calculated for the target fragments by tau-ratio method. The bend angles of four of the target fragments were independent of temperature from 4 degrees C to 20 degrees C, but decreased when the temperature was increased to 37 degrees C. The bend angles of the other two target fragments were independent of temperature over the entire range examined, 4 degrees -37 degrees C. Hence, the thermal stability of sequence-dependent bends in random-sequence DNA is variable. The bend angles of five of the six target fragments were independent of the presence or absence of Mg2+ ions in the solution, indicating most of the target fragments were stably bent or curved, rather than anisometrically flexible. Restriction fragments containing 219 and 224 bp, with sequences somewhat offset from the sequence of the 207 bp fragment, were also studied. Comparison of the tau-ratios of these overlapping fragments allowed both the bend angle and bend position to be independently determined. These methods should be useful for analyzing sequence-dependent bending in other random-sequence DNAs.

Biophysical Phenomena↗
PubMed2003

Free solution mobility of small single-stranded oligonucleotides with variable charge densities.

The free solution mobilities of six single-stranded 16-nucleotide DNA oligomers with the same sequence, containing up to 11 neutral phosphoramidate internucleoside linkages, have been measured by capillary electrophoresis. The mobilities of the partially charged oligomers increase linearly with the logarithm of increasing charge density, as predicted by the Manning theory of electrophoresis (G. S. Manning, J. Phys. Chem. 1981, 85, 1506-1515). For comparison, the mobilities of eight fully charged single-stranded oligomers containing similar numbers of charged phosphate residues have also been measured. The mobilities of the variable length, fully charged oligomers increase more rapidly with the increasing number of charged phosphate residues than the mobilities of the constant size, partially charged phosphoramidate derivatives, because of the larger diffusion coefficients of the modified oligomers.

Amides↗
PubMed2003

Sequence-dependent bending in plasmid pUC19.

The circular permutation assay has been used to characterize sequence-dependent bending in plasmid pUC19 (2686 bp). Linear, permuted sequence isomers of pUC19 exhibit different mobilities in large-pore polyacrylamide gels, suggesting that the plasmid contains two sequence-dependent bends, one located at approximately 806 bp, close to the start site of transcription, and the other at approximately 2617 bp, near the promoter of the ampicillin resistance gene. The mobility patterns are independent of the buffer in which the gels are cast and run, independent of pH over the range 6.5-8.6, and independent of the presence or absence of various monovalent ions added to the buffer, suggesting that the maxima in the mobility patterns correspond to stable bends in the helix backbone, not anisometric flexibility. Although the mobility patterns are unchanged when Ba(++) or Zn(++) ions are added to the buffer, several of the sequence isomers exhibit multiple sharp sub-bands in solutions containing Mg(++) or Ca(++) ions, indicating the presence of multiple conformational isomers. In addition, the mobility maximum near the start site of transcription is shifted by approximately 100 bp in the presence of Ca(++) ions, suggesting that the location of the actual bend center is somewhat variable, depending on the ionic composition of the surrounding medium. The number of maxima observed in the mobility pattern can be increased by adding a bend from another plasmid, such as Litmus 28; the number of maxima can be decreased by removing one of the bends in pUC19 by restriction enzyme digestion and religation. Hence, the number of maxima in the mobility pattern is an accurate indication of the number of stable, sequence-dependent bends present in the plasmid. The combined results indicate that the circular permutation assay in large-pore polyacrylamide gels is a reliable method of detecting and analyzing sequence-dependent bends in kilobase-sized DNA molecules.

Base Sequence↗
PubMed2003

Analysis of the intrinsic bend in the M13 origin of replication by atomic force microscopy.

Atomic force microscopy (AFM) has been used to image a 471-bp bent DNA restriction fragment derived from the M13 origin of replication in plasmid LITMUS 28, and a 476-bp normal, unbent fragment from plasmid pUC19. The most probable angle of curvature of the 471-bp DNA fragment is 40-50 degrees, in reasonably good agreement with the bend angle determined by transient electric birefringence, 38 degrees +/- 7 degrees. The normal 476-bp DNA fragment exhibited a Gaussian distribution of bend angles centered at 0 degrees, indicating that this fragment does not contain an intrinsic bend. The persistence length, P, was estimated to be 60 +/- 8 and 62 +/- 8 nm for the 471- and 476-bp fragments, respectively, from the observed mean-square end-to-end distances in the AFM images. Since the P-values of the normal and bent fragments are close to each other, the overall flexibility of DNA fragments of this size is only marginally affected by the presence of a stable bend. The close agreement of AFM and transient electric birefringence results validates the suitability of both methods for characterizing DNA bending and flexibility.

Crystallography↗
PubMed2003

Probing the electrostatic shielding of DNA with capillary electrophoresis.

The free solution mobility of a 20-bp double-stranded DNA oligomer has been measured in diethylmalonate (DM) and Tris-acetate buffers, with and without added NaCl or TrisCl. DM buffers have the advantage that the buffering ion is anionic, so the cation composition in the solution can be varied at will. The results indicate that the free solution mobility of DNA decreases linearly with the logarithm of ionic strength when the ionic strength is increased by increasing the buffer concentration. The mobility also decreases linearly with the logarithm of ionic strength when NaCl is added to NaDM buffer or TrisCl is added to TrisDM buffer. Nonlinear effects are observed if the counterion in the added salt differs from the counterion in the buffer. The dependence of the mobility on ionic strength cannot be predicted using the Henry, Debye-Hückel-Onsager, or Pitts equations for electrophoresis. However, the mobilities observed in all buffer and buffer/salt solutions can be predicted within approximately 20% by the Manning equation for electrophoresis, using no adjustable parameters. The results suggest that the electrostatic shielding of DNA is determined not only by the relative concentrations of the various ions in the solution, but also by their equivalent conductivities.

Computer Simulation↗
PubMed2002

The free solution mobility of DNA in Tris-acetate-EDTA buffers of different concentrations, with and without added NaCl.

The free solution mobility of a high-molecular-weight DNA, linear pUC19, and a 20-bp oligomer called dsA5 have been studied as a function of Tris-acetate-EDTA (TAE) buffer concentration, with and without added NaCl. The two DNAs migrate as separate peaks during capillary electrophoresis, because the mobility of linear pUC19 is higher than that of the 20-bp oligomer. In TAE buffers ranging from 10-400 mM in concentration, the migration times and peak areas of the two DNAs are independent of whether they are electrophoresed separately or in mixtures, indicating that DNA-DNA and DNA-buffer interactions are absent in these solutions. The migration times of the two DNAs vary and the peak areas are not additive when the TAE buffer concentration is reduced to 5 mM or below, indicating that DNA-DNA and DNA-buffer interactions are occurring at very low TAE buffer concentrations. The mobilities of linear pUC19 and dsA5 decrease slowly with increasing conductivity or ionic strength when the conductivity is increased by increasing the TAE buffer concentration. When the Tris buffer concentration is held constant and the conductivity is increased by adding various concentrations of NaCl to the solution, the mobilities of linear pUC19 and dsA5 first increase slightly, then become independent of solution conductivity (or ionic strength), and finally decrease when the NaCl concentration is increased above approximately 50 mM. The mobility variations observed in the various TAE and TAE-NaCl solutions are described qualitatively by Manning's theory, although quantitative agreement is not achieved. The free solution mobilities of single-stranded pUC19 and two 20-base oligonucleotides have also been measured. The free solution mobility of single-stranded pUC19 is approximately 15% lower than that of native pUC19, in agreement with other results in the literature. Somewhat surprisingly, the mobilities of the single- and double-stranded 20-mers are equal to each other in TAE buffers with and without added NaCl.

Acetates↗
PubMed2002

Modeling the gel electrophoresis of short duplex DNA by Brownian dynamics: cubic gel lattice with direct interaction.

The technique of Brownian dynamics is used to model the electrophoretic mobility of spherical and rod-like particles in a three-dimensional cubic gel lattice. In addition to excluded volume interactions between the migrating particle and the gel, direct interactions are also included. The methodology is first applied to spherical particles in the absence of direct interactions and the resulting mobilities are shown to agree with independent studies. The methodology is then applied to rod-like models of short duplex DNA fragments 10-50 base pairs in length. In the absence of direct interactions between gel and DNA, calculated mobilities show a much weaker dependence on gel concentration than observed in experiments of DNA in Tris-acetate buffer and polyacrylamide gels. When an attractive interaction between gel and DNA of approximately -0.3 k(B)T per base pair at contact is included, good agreement between calculated and experimental mobilities is achieved.

DNA↗
PubMed2002

Determining the electrophoretic mobility and translational diffusion coefficients of DNA molecules in free solution.

The free solution mobility of DNA molecules of different molecular weights, the sequence dependence of the mobility, and the diffusion coefficients of small single- and double-stranded DNA (ss- and dsDNA) molecules can be measured accurately by capillary zone electrophoresis, using coated capillaries to minimize the electroosmotic flow (EOF) of the solvent. Very small differences in mobility between various analytes can be quantified if a mobility marker is used to correct for small differences in EOF between successive experiments. Using mobility markers, the molecular weight at which the free solution mobility of dsDNA becomes independent of molecular weight is found to be approximately 170 bp in 40 mM Tris-acetate-EDTA buffer. A DNA fragment containing 170 bp has a contour length of approximately 58 nm, close to the persistence length of DNA under these buffer conditions. Hence, the approach of the free solution mobility of DNA to a plateau value may be associated with the transition from a rod-like to a coil-like conformation in solution. Markers have also been used to determine that the free solution mobilities of ss- and dsDNA oligomers are sequence-dependent. Double-stranded 20-bp oligomers containing runs of three or more adenine residues in a row (A-tracts) migrate somewhat more slowly than 20-mers without A-tracts, suggesting that somewhat larger numbers of counterions are condensed in the ion atmospheres of A-tract DNAs, decreasing their net effective charge. Single-stranded 20-mers with symmetric sequences migrate approximately 1% faster than their double-stranded counterparts, and faster than single-stranded 20-mers containing A(5)- or T(5)-tracts. Interestingly, the average mobility of two complementary single-stranded 20-mers is equal to the mobility of the double-stranded oligomer formed upon annealing. Finally, the stopped migration method has been used to measure the diffusion coefficients of single- and double-stranded oligomers. The diffusion coefficients of ssDNA oligomers containing 20 nucleotides are approximately 50% larger than those of double-stranded DNA oligomers of the same size, reflecting the greater flexibility of ssDNA molecules. The methods used to carry out these experiments are also described in detail.

Base Sequence↗