Highly sensitive electrochemiluminescence displacement method for the study of DNA/small molecule binding interactions.
Non-covalent binding interactions of small molecules with DNA play important roles in regulating gene expression and gene function. In this work, a highly sensitive electrochemiluminescence (ECL) displacement method has been developed to investigate such interactions, particularly for weak DNA binders. This ECL method relies on a double-stranded DNA film deposited on an indium tin oxide electrode (ITO) surface by layer-by-layer self-assembly. A DNA intercalator, [Ru(bpy)(2)(dppz)](2+) (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2'3'-c]phenazine), is employed as the ECL signal indicator. If a test compound competes with the indicator for the same binding sites in DNA, it would displace the indicator from the film and reduce ECL signal. The new method was validated by measuring five well-known DNA-binding organic molecules including quinacrine, H33258, thiazole orange, ethidium bromide and 4,6-diamidine-2-phenylindole dihydrochloride. Due to high ECL sensitivity, only 0.4 micromol L(-1) [Ru(bpy)(2)(dppz)](2+) was needed in the ECL displacement measurement, which is about 75-fold less than the concentration in the voltammetric measurement. The lowered concentration permitted direct measurement of IC(50) values of eight hydroxylated polycyclic aromatic hydrocarbons in their ECL displacement curves and subsequent calculation of their binding constants with DNA. The ECL displacement method is particularly useful for investigating weak DNA binders with limited aqueous solubility.
Huang
Rongfu
R
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, P.O. Box 2871, Beijing 100085, China.
Wang
Li-Rong
LR
Guo
Liang-Hong
LH
D004247:DNA / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000379:methods*; D004566:Electrodes; D007364:Intercalating Agents / Q000737:chemistry; D008024:Ligands; D008163:Luminescent Measurements / Q000379:methods*; D009942:Organometallic Compounds / Q000737:chemistry; D010619:Phenazines / Q000737:chemistry; D017971:Tin Compounds / Q000737:chemistry
D007364:Intercalating Agents; D008024:Ligands; D009942:Organometallic Compounds; D010619:Phenazines; D017971:Tin Compounds; C109984:indium tin oxide; C077849:bis(2,2'-bipyridyl)(dipyrido(3,2-alpha-2',3'-c)phenazine)ruthenium (II); D004247:DNA