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The thermal stabilities of DNA duplexes analogous to the let-7 microRNA: lin-41 mRNA complex from Caenorhabditis elegans have been measured by free solution capillary electrophoresis. DNA duplexes with the same stems but different types of internal or bulge loops and a control with no loop have also been studied. The melting temperatures of the DNA derivatives increased linearly with the logarithm of the Na + or K + ion concentration in the solution. Peaks in the electropherograms corresponding to duplexes with internal or bulge loops exhibited extensive tailing at high temperatures, suggesting that denaturation occurred by slow exchange between the duplexes and their component single strands. The single strands did not separate completely from the duplexes in aqueous solutions; instead, they appeared as small subpeaks on the tails of the duplex peaks. However, complete separation of the duplexes from their component single strands was observed at 20 °C in solutions containing 300 mM tetrapropylammonium ions. In addition, counterion condensation appears to be significantly reduced in DNA duplexes containing internal or bulge loops.

The free-solution mobilities of small single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) have been measured by capillary electrophoresis in solutions containing 0.01-1.0 M sodium acetate. The mobility of dsDNA is greater than that of ssDNA at all ionic strengths because of the greater charge density of dsDNA. The mobilities of both ssDNA and dsDNA decrease with increasing ionic strength until approaching plateau values at ionic strengths greater than ∼0.6 M. Hence, ssDNA and dsDNA appear to interact in a similar manner with the ions in the background electrolyte. For dsDNA, the mobilities predicted by the Manning electrophoresis equation are reasonably close to the observed mobilities, using no adjustable parameters, if the average distance between phosphate residues (the b parameter) is taken to be 1.7 Å. For ssDNA, the predicted mobilities are close to the observed mobilities at ionic strengths ≤0.01 M if the b-value is taken to be 4.1 Å. The predicted and observed mobilities diverge strongly at higher ionic strengths unless the b-value is reduced significantly. The results suggest that ssDNA strands exist as an ensemble of relatively compact conformations at high ionic strengths, with b-values corresponding to the relatively short phosphate-phosphate distances through space., (Copyright © 2020. Published by Elsevier Inc.)

Capillary electrophoresis has been used to measure the thermal stability of small DNA hairpins in solutions containing 0.3 M cation, comparing the results observed in Na + and NH 4 + with those observed in solutions containing various tetraalkylammonium ions. The midpoint melting temperatures of the hairpins decreased nonlinearly with cation radius but linearly with solvent viscosity, suggesting that the reversible melting transition involves DNA migration through the solvent to find stable base-pairing partners. The normalized melting temperatures increased linearly with the inverse viscosity of the solvent and agreed with values calculated from literature data for another small DNA hairpin, a small RNA duplex, and sonicated calf thymus DNA in tetraalkylammonium ion solutions. The normalized melting temperatures calculated from literature data for poly(A)·poly(U) and two proteins, ribonuclease and lysozyme, in tetraalkylammonium ion solutions also increased linearly with inverse solvent viscosity. By contrast, the normalized melting temperatures calculated from literature data for DNA in solutions containing ethylene glycol or glycerol to modify the viscosity increased linearly with the logarithm of inverse solvent viscosity, not the first power of inverse solvent viscosity.

Free solution electrophoretic mobilities of polyelectrolytes with different charge densities have been analyzed using data taken from the literature. The polyions include single- and double-stranded DNA oligomers, small aromatic molecules, peptides, proteins, and synthetic copolymers. Mobility variations due to differences in the background electrolytes were minimized by calculating mobility ratios, dividing the mobility of each charge variant in each data set by the mobility of the most highly charged polyion in that data set. In all cases, the mobility ratios increase linearly with the logarithm of the fractional charge, not the first power of the charge as usually assumed. In addition, the mobility ratios observed for all polyelectrolytes, except for the synthetic copolymers, exhibit a common dependence on the logarithm of fractional charge. The unique results observed for the synthetic copolymers may be due to the flexibility of their hydrocarbon backbones, in contrast to the relatively rigid hydrophilic backbones of the other polyelectrolytes. The mobilities observed for the DNA charge variants are well predicted by the Manning electrophoresis equation, whereas the mobilities predicted by zeta potential theories are higher. However, mobility ratios calculated from both theories agree with the observed results.

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(+)) 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(+) and slower than the reference in Li(+). Hence, cations of different sizes can interact very differently with DNA A-tracts. The increased mobilities observed in TBA(+) suggest that the large hydrophobic TBA(+) 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(+) ions decrease the mobility of A-tract oligomers because of the preferential localization of Li(+) 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., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Monovalent cation binding by DNA A-tracts, runs of four or more contiguous adenine or thymine residues, has been determined for two curved approximately 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 degrees and 46 degrees , respectively, located within approximately 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.

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.

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.

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.

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.

The technique of transient electric birefringence was used to investigate the effect of pulsed electric fields on the orientation of the agarose gel matrix. Orientation of the gel was observed at all electric field strengths. Very slow, time-dependent effects were observed when pulses of 10-100 V/cm were applied to 1% gels for 0.5-2 seconds, indicating that domains of the matrix were being oriented by the electric field. The sign of the birefringence reversed when the direction of the applied electric field was reversed, indicating that the domains tend to orient in the perpendicular direction after field reversal. Theories of gel electrophoresis will need to incorporate the orientation of the matrix in order to provide a complete explanation of electrophoresis in agarose gels.

It was found that the birefringence of aqueous solutions of sodium DNA is anomalous when electric fields of high intensity (>/=10(4) v/cm) are applied. The magnitude of the birefringence first rose upon application of the orienting pulse, then fell as the field was sustained above a critical value. The occurrence of the effect depended upon macromolecular and electrolyte concentrations. Upon removal of the field, the birefringence was rapidly restored and then it decayed with an increase of the reorientational relaxation times, relative to those observed below the critical field. It is proposed that the electric field may cause aggregation of the macromolecules and then produce a structural transition concomitant with the electric field orientation effect. This transition may correspond to the "B" right harpoon over left harpoon "A" structures identified in x-ray studies, or to a "B" right harpoon over left harpoon "V" structure change, where "V" is a postulated new helical form stabilized by cooperative interactions of base and dipoles in the electric field. Field induced transitions of this type would be of interest in connection with molecular mechanisms of transport through membranes, nerve impulse transmission, or information storage.