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

Abstract

Using capillary electrophoresis to characterize the hydrodynamic and electrostatic properties of DNA in solutions containing various monovalent cationsThis 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. The mobilities of both ss-and dsDNAs increase linearly with the logarithm of increasing charge per unit length and decrease linearly with the logarithm of increasing ionic strength. Surprisingly, ss-and dsDNA mobilities level off and become nearly constant at ionic strengths ≥0.6 M. The thermal stabilities of dsDNAs decrease linearly with increasing solution viscosity. The diffusion coefficients of dsDNA are modulated by the diffusion coefficients of their counterions because of electrostatic DNA-cation coupling interactions. Finally, the anomalously slow mobilities observed for A-tract-containing DNAs can be attributed both to differences in shape and to the preferential localization of small cations in the A-tract minor groove. Since many of these results are mirrored in other polyion-counterion systems, free-solution electrophoresis can be viewed as a reporter of the electrostatics and hydrodynamics of highly charged polyions. New results describing the mobilities of dsDNA analogues of a microRNA-messenger RNA complex are also presented.

Monovalent cation localization in DNA A‐tracts with different sequences

“…A large, presumably nonbinding ion such as the TPA + , is gradually replaced by a potential binding ion, such as Li + , keeping the total ionic strength of the solution constant. The constant ionic strength prevents the large variation in mobility with ionic strength [23,32] from obscuring smaller changes due to monovalent cation localization in DNA A-tracts [33,36]. The TPA + ions also allowed the CE measurements to be carried out at lower [Li + ] than possible in solutions containing Li + ions alone.…”

“…Importantly, the translational diffusion coefficients of DNA oligomers containing the same number of base pairs are independent of the effective charge of the oligomer and, hence, independent of monovalent cation localization in the A-tract minor groove [22,25,26]. As the free solution mobility of a polyion is determined by the ratio of its effective charge to its translational diffusion coefficient [27][28][29][30][31][32], a decrease in the mobility of an A-tract-containing oligomer in background electrolytes (BGEs) containing small monovalent cations is an unambiguous indicator of monovalent cation localization in the A-tract minor groove [15,22,24].…”

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

“…The free solution mobility of an electrolyte such as DNA is determined by the ratio between its effective charge and its frictional coefficient [27,29,[34][35][36][37][38][39][40][41]. If the EOF of the solvent is negligible, the mobility can be calculated directly from the observed migration times.…”

“…The mobility ratios were then plotted as a function of the concentration of the small monovalent cation in the BGE; such plots are called mobility ratio profiles for brevity. Importantly, the constant ionic strength of the BGE prevented the large variation in mobility with ionic strength [26,29] from obscuring the smaller changes in mobility due to monovalent cation localization in the A-tract minor groove [27][28][29]33]. Transitions observed in the mobility ratio profiles were analyzed by fitting the mobility ratios to four-parameter sigmoidal decays using the fitting programs in SigmaPlot.…”

“…CE is a good reporter of monovalent cation localization in the A-tract minor groove because the electrophoretic mobilities of oligomers containing the same number of bps are independent of sequence [25][26][27]. However, if one of the oligomers contains an A-tract that localizes monovalent cations in the A-tract minor groove, the effective charge of the two oligomers will differ and the mobilities of the A-tract and non-A-tract oligomers will be different [28,29]. In addition, we have shown that the affinity for monovalent cation localization in the A-tract minor groove is cation-specific [27] and can be blocked if the A-tract had previously been treated with netropsin, a high-affinity A-tract minor groove-binding drug [30,31].…”