Effect of Internal and Bulge Loops on the Thermal Stability of Small DNA Duplexes.

The thermal stabilities of DNA duplexes analogous to the microRNA: mRNA complex from 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.

Flanking AT base pairs affect the localization of monovalent cations in DNA A-tracts.

Capillary electrophoresis has been used to measure the free solution mobilities of a series of 26-base pair (bp) DNA oligomers containing two phased A4T1in-tracts embedded in flanking sequences containing 0 to 11 additional AT bps. A random-sequence 26-bp oligomer with 12 isolated AT bps was used as the reference. Mobility ratios (A-tract/reference) were measured in background electrolytes (BGEs) containing mixtures of small monovalent cations and tetrabutylammonium (TBA ) or tetrapropylammonium (TPA ) ions.

Monovalent cation localization in DNA A-tracts with different sequences.

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.

Using capillary electrophoresis to characterize the hydrodynamic and electrostatic properties of DNA in solutions containing various monovalent cations.

This review describes the results obtained by using free-solution capillary electrophoresis to probe the electrostatic and hydrodynamic properties of DNA in solutions containing various monovalent cations. In brief, we found that the mobilities of double-stranded DNAs (dsDNAs) increase with increasing molecular weight before leveling off and becoming constant at molecular weights ≥400 bp. The mobilities of single-stranded DNAs (ssDNAs) go through a maximum at ∼10-20 nucleotides before decreasing and becoming constant for oligomers larger than ∼30-50 bases.

Electrophoretic Mobility of DNA in Solutions of High Ionic Strength.

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.

DNA Thermal Stability Depends on Solvent Viscosity.

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 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.

Electrophoretic Mobilities of the Charge Variants of DNA and Other Polyelectrolytes: Similarities, Differences, and Comparison with Theory.

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.

Electrostatic coupling between DNA and its counterions modulates the observed translational diffusion coefficients.

Free solution capillary electrophoresis (CE) is a useful technique for measuring the translational diffusion coefficients of charged analytes. The measurements are relatively fast if the polarity of the electric field is reversed to drive the analyte back and forth past the detection window during each run. We have tested the validity of the resulting diffusion coefficients using double-stranded DNA molecules ranging in size from 20 to 960 base pairs as the model system.

Flanking A·T basepairs destabilize the B(∗) conformation of DNA A-tracts.

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.

The free solution mobility of DNA and other analytes varies as the logarithm of the fractional negative charge.

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.

DNA A-tracts are not curved in solutions containing high concentrations of monovalent cations.

The intrinsic curvature of seven 98 bp DNA molecules containing up to four centrally located A6-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.

Tandem GA residues on opposite sides of the loop in molecular beacon-like DNA hairpins compact the loop and increase hairpin stability.

The free solution electrophoretic mobilities and thermal stabilities of hairpins formed by two complementary 26-nucleotide oligomers have been measured by capillary electrophoresis. The oligomers are predicted to form molecular beacon-like hairpins with 5 bp stems and 16 nucleotides in the loop. One hairpin, called hairpin2 (hp2), migrates with a relatively fast free solution mobility and exhibits melting temperatures that are reasonably well predicted by the popular structure-prediction program Mfold.

Monovalent cation size and DNA conformational stability.

The effect of monovalent cations on the thermal stability of a small model DNA hairpin has been measured by capillary electrophoresis, using an oligomer with 16 thymine residues as an unstructured control. The melting temperature of the model hairpin increases approximately linearly with the logarithm of increasing cation concentration in solutions containing Na(+), K(+), Li(+), NH(4)(+), Tris(+), tetramethylammonium (TMA(+)), or tetraethylammonium (TEA(+)) ions, is approximately independent of cation concentration in solutions containing tetrapropylammonium (TPA(+)) ions, and decreases with the logarithm of increasing cation concentration in solutions containing tetrabutylammonium (TBA(+)) ions. At constant cation concentration, the melting temperature of the DNA model hairpin decreases in the order Li(+) ∼ Na(+) ∼ K(+) > NH(4)(+) > TMA(+) > Tris(+) > TEA(+) > TPA(+) > TBA(+).

Effect of magnesium ions and temperature on the sequence-dependent curvature of DNA restriction fragments.

Transient electric birefringence has been used to quantify the curvature of two DNA restriction fragments, a 199-base-pair fragment taken from the origin of replication of the M13 bacteriophage and a 207-base-pair fragment taken from the VP1 gene in the SV40 minichromosome. Stable curvature in the SV40 and M13 restriction fragments is due to a series of closely spaced A tracts, runs of 4-6 contiguous adenine residues located within 40 or 60 base pair 'curvature modules' near the center of each fragment. The M13 and SV40 restriction fragments exhibit bends of ∼ 45° in solutions containing monovalent cations and ∼ 60° in solutions containing Mg(2 +) ions.

Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution.

This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels.

Monovalent cation binding in the minor groove of DNA A-tracts.

The binding of five different monovalent cations to DNA oligomers containing A-tracts, runs of four or more contiguous adenine residues, has been assessed by capillary electrophoresis, using the Replacement Ion method. In this method, a nonbinding 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 oligomer, decreasing its free solution mobility.

Effect of the matrix on DNA electrophoretic mobility.

DNA electrophoretic mobilities are highly dependent on the nature of the matrix in which the separation takes place. This review describes the effect of the matrix on DNA separations in agarose gels, polyacrylamide gels and solutions containing entangled linear polymers, correlating the electrophoretic mobilities with information obtained from other types of studies. DNA mobilities in various sieving media are determined by the interplay of three factors: the relative size of the DNA molecule with respect to the effective pore size of the matrix, the effect of the electric field on the matrix, and specific interactions of DNA with the matrix during electrophoresis.

Do zwitterions contribute to the ionic strength of a solution?

Capillary electrophoresis has been used to determine whether zwitterions contribute to the ionic strength of a solution, by measuring the mobility of a double-stranded DNA oligomer in cacodylate-buffered solutions containing various concentrations of the ionic salt tetraethylammonium chloride (TEA(+)Cl(-)) or the zwitterion tricine(+/-). The mobility of the DNA decreased as the square root of ionic strength, as expected from the Debye-Hückel-Onsager theory of electrophoresis, when TEA(+)Cl(-) was added to the buffer. However, the mobility was independent of the concentration of added tricine(+/-).

Monovalent cation binding by curved DNA molecules containing variable numbers of a-tracts.

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.

Electrophoretic mobility is a reporter of hairpin structure in single-stranded DNA oligomers.

The electrophoretic mobilities of 24 single-stranded DNA oligomers, each containing 26 nucleotide residues, have been measured in polyacrylamide gels and in free solution. The mobilities observed at 20 degrees C differed by approximately 20% in polyacrylamide gels and by approximately 10% in free solution, even though the oligomers contained the same number of bases. Increasing the temperature or adding urea to the solution equalized the mobilities of the oligomers, suggesting that the variable mobilities observed at 20 degrees C are due to the formation of stable secondary structures, most likely hairpins.

Capillary electrophoresis is a sensitive monitor of the hairpin-random coil transition in DNA oligomers.

Capillary electrophoresis (CE) has been used to characterize the hairpin-random coil transition of four octamers in the GCxxxxGC minihairpin family, where xxxx is GAAA, TTTC, TTTT, or AAAA. The transition can be monitored by CE because differences in the frictional coefficients of the hairpin and coil forms of each octamer lead to a difference of approximately 9% in the free solution mobilities of the two conformations. The GAAA octamer is unusually stable, with a melting temperature of 65 degrees C.

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.

The free solution mobilities of the adenosine nucleotides 5'-adenosine triphosphate (ATP), 5'-adenosine diphosphate (ADP), 5'-adenosine monophosphate (AMP), and 3'-5'-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.

Quantitative analysis of monovalent counterion binding to random-sequence, double-stranded DNA using the replacement ion method.

A variation of affinity capillary electrophoresis, called the replacement ion (RI) method, has been developed to measure the binding of monovalent cations to random sequence, double-stranded (ds) DNA. In this method, the ionic strength is kept constant by gradually replacing a non-binding ion in the solution with a binding ion and measuring the mobility of binding and non-binding analytes as a function of binding ion concentration. The method was validated by measuring the binding of Li+ ions to adenosine nucleotides; the apparent dissociation constants obtained by the RI method are comparable to literature values obtained by other methods.

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.

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.