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. The binding of Tris+, NH4+, Li+, Na+, and K+ to dsDNA was then investigated. The apparent dissociation constants observed for counterion binding to a random-sequence 26-base pair (bp) oligomer ranged from 71 mM for Tris+ to 173 mM for Na+ and K+. Hence, positively charged Tris buffer ions will compete with other monovalent cations in Tris-buffered solutions. The bound cations identified in this study may correspond to the strongly correlated, tightly bound ions recently postulated to exist as a class of ions near the surface of dsDNA (Tan, Z.-J., and Chen, S.-J. (2006) Biophys. J. 91, 518-536). Monovalent cation binding to random-sequence dsDNA would be expected to occur in addition to any site-specific binding of cations to A-tracts or other DNA sequence motifs. Single-stranded DNA oligomers do not bind the five tested cations under the conditions investigated here.
Stellwagen
Earle
E
Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA.
Dong
Qian
Q
Stellwagen
Nancy C
NC
D002021:Buffers; D002414:Cations, Monovalent / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods; D008094:Lithium / Q000737:chemistry; D009994:Osmolar Concentration; D011188:Potassium / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D014325:Tromethamine / Q000737:chemistry
D002021:Buffers; D002414:Cations, Monovalent; D000644:Quaternary Ammonium Compounds; D014325:Tromethamine; D004247:DNA; D008094:Lithium; D012964:Sodium; D011188:Potassium