Ionic Mobilities of Duplex and Frayed Wire DNA in Discontinuous Buffer Electrophoresis: Evidence of Interactions with Amino Acids
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abstract
Nucleic acid-amino acid interactions are fundamental to understanding higher-order interactions made by nucleic acid-binding proteins. Here we employ electrophoresis to investigate DNA-amino acid interactions by using a set of amino acids (Ala, Gln, Gly, Met, Phe, Val, bicine and tricine) as trailing ions in a discontinuous buffer, and monitoring their interactions with duplex (from 12 to 3000 bp) and frayed wire [a set of self-assembled superstructures arising from d(A(15)G(15)) oligodeoxyribonucleotides] DNA by the change in their ionic mobility (in terms of %R(f)) as a function of amino acid concentration in a polyacrylamide matrix. By titration of the pH of Tris-HCl polyacrylamide gels (from 7 to 10), a span of steady-state amino acid concentrations and extents of ionization can be maintained. We found that with a decrease in pH (thereby increasing amino acid concentrations and the extent of ionization of the alpha-amino group), both the %R(f) and stacking limit were increased, but the extent varied among the trailing ions, resulting in an induced dispersion of %R(f) values for a given analyte. Using singular-value analysis to take into account the %R(f) dependence on fragment size (i.e., the %R(f) distribution), the degree of dispersion was found to be positively correlated with the accumulation of N-protonated trailing ions in the resolving phase. These results indicate that the modification of %R(f) of DNA is a mass-action effect involving DNA-amino acid interactions under essentially aqueous conditions.
