Study of interactions between actinomycin D and oligonucleotides by microchip electrophoresis and ESI-MS

In the present study, the interactions between actinomycin D (ActD) and single stranded DNA (ssDNA) 5'-CGTAACCAACTGCAACGT-3' and a duplex stranded DNA (dsDNA) with this sequence were investigated by microchip-based non-gel sieving electrophoresis and electrospray ionization mass spectrometry (ESI-MS). The ssDNA was designed according to the conserved regions of open reading frame 1b (replicase 1B) following the Tor 2 SARS genome sequence of 15611-15593. The binding constants of the interactions between ActD and ssDNA/dsDNA were (8.3+/-0.32)x10(6)M(-1) (ssDNA) and (2.8+/-0.02)x10(5)M(-1) (dsDNA), respectively, calculated from microchip electrophoresis via Scatchard plot. The binding stoichiometries were 1:1 (single/1ActD molecule) and 1:2 (duplex/2ActD molecules) calculated from microchip electrophoresis, and the results were further verified by ESI-MS. The results obtained by these two methods indicated that ActD bound much more tightly to ssDNA used in this work than dsDNA. Furthermore, this is shown that the microchip-based non-gel sieving electrophoresis method is a rapid, highly sensitive and convenient method for the studies of interactions between DNA and small molecule drugs.