Transient association of the DNA-ligand complex during gel electrophoresis.
DNA frayed wires are extremely stable multistranded complexes arising from the association of oligonucleotides with long terminal runs of consecutive guanines. Frayed wires originating from d(A15G15) have multiple binding sites for short complementary oligonucleotides such as dT10. We examine unusual band patterns obtained when complexes formed between dT10 and DNA frayed wires are resolved on nondenaturing polyacrylamide gels. Since the lifetime of the dT10-frayed wire complexes is shorter than the time of the gel run, the interaction between the components during the gel electrophoresis affects their band patterns. We have conducted chasing experiments to show that (i) the binding of dT10 to the frayed wires can occur during gel electrophoresis, and (ii) dissociation of the complexes occurs during the gel run. Rapid repetitive dissociation-reassociation of the complexes leads to a constant partitioning of dT10 between their binding sites within frayed wires. Consequently, complexes composed of frayed wires and various numbers of bound ligands appear on the gel as a single well-defined band. The mobilities of these bands decrease continuously with the concentration of the ligand reaching saturation when all the binding sites are occupied. This characteristic pattern is observed only for relatively unstable interactions. Longer ligands, i.e., oligonucleotides with higher affinity towards the binding sites, cease to exhibit the dynamic character of interaction during gel electrophoresis. These ligands form long-lived complexes with the frayed wires that appear on the gel as faint smeared bands reflecting the presence of multiple stable complexes.
Protozanova
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Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, ON, Canada.
Macgregor
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RB
D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer; D006147:Guanine / Q000378:metabolism; D006358:Hot Temperature; D008024:Ligands; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011071:Poly T / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011071:Poly T; D006147:Guanine; D004247:DNA