Determination of the Binding Constant between Oligonucleotide-Coupled Magnetic Microspheres and Target DNA

Oligonucleotide-coupled magnetic microspheres (PINIPs) are commonly used as solid carriers in genetic analysis to specifically recognize, capture, and manipulate target DNA. Determining the binding constant (K-A) of nucleic acid hybridization using an assay based on magnetic microspheres (MPs) and evaluating the performance of magnetic microspheres as solid carriers has significance for many applications. In this work, we established a simple double-reciprocal plot method to determine the binding constant of nucleic acid hybridization based on magnetic microspheres. The main experimental parameters were first optimized, and the binding constants between PMPs with single-stranded DNA (ssDNA) and PMPs with double-stranded DNA (dsDNA) were 1.07 +/- 0.01 X 10(8) and 0.77 +/- 0.02 X 10(8) M-1, respectively, through the established double-reciprocal plot method. Oligonucleotide-coupled magnetic microspheres have the same initial effective target binding sites (A(0)), regardless of whether the target molecules are single-stranded or double-stranded. In addition, the binding constants between PMPs with ssDNA and PMPs with dsDNA were 18.58 and 13.37%, respectively, of the binding constants measured in solution by isothermal titration calorimetry. This finding indicates that the binding constant based on solid phase hybridization is not significantly lower than that of liquid phase hybridization, even in the presence of competing target nucleic acid molecules in solution.