Ionic mobilities of duplex and frayed wire DNA in discontinuous buffer electrophoresis: evidence of interactions with amino acids.
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.
Poon
Gregory M K
GM
Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada M5S 2S2.
Abu-Ghazalah
Rashid M
RM
Macgregor
Robert B
RB
D000596:Amino Acids / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D003627:Data Interpretation, Statistical; D004586:Electrophoresis; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000378:metabolism
D000596:Amino Acids; D007477:Ions; D004247:DNA