A surface plasmon resonance method for detecting multiple modes of DNA-ligand interactions.
A simple and general surface plasmon resonance (SPR) based method has been developed to detect and quantitate binding of low molecular weight compounds (200-1,200 Da) to double stranded DNA. Several compounds were chosen to probe three different modes of binding interactions, intercalation, minor groove binding and electrostatic interactions. Ethidium bromide (MW 390 Da), a probe of intercalative binding, was tested by plotting the steady state SPR responses measured on a DNA modified surface versus ethidium bromide concentration. The best fit of the binding isotherm gave a Keq of 1.8 x 105 M-1. Co-solvents such as DMSO are often used in activity assays to increase the solubility of poorly water-soluble drugs. The effect of DMSO on the ethidium bromide/DNA interaction was also tested by measuring binding in the presence of 0, 1 and 5%, DMSO. No effect on the measured Keq was observed at these DMSO concentrations. The binding of actinomycin (MW 1,255 Da), an antibiotic known to bind DNA through intercalation and minor groove binding, was also tested. The Keq estimated from the steady state responses on a DNA surface was 1.9 x 106 M-1. DAPI (MW 350 Da) (4',6-diamidino-2-phenylindole) a fluorescent probe which binds the minor groove of DNA was also tested and gave a Keq of 1.8 x 106 M-1 measured by SPR. Finally, spermine (MW 202) a compound known to bind DNA through ionic interactions gave the weakest Keq of 1.7 x 104 M-1. All the Keq values measured by SPR and reported for these compounds were in good agreement with literature values measured by other techniques.
Ciolkowski
M L
ML
Pharmaceutical Development, Pharmacia and Upjohn Inc, Kalamazoo. MI 49007-4940, USA. mary.l.ciolkowski@am.pnu.com
Fang
M M
MM
Lund
M E
ME
D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000737:chemistry; D003609:Dactinomycin / Q000737:chemistry; D004996:Ethidium / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D007364:Intercalating Agents / Q000737:chemistry; D008024:Ligands; D009841:Oligonucleotides / Q000737:chemistry; D013096:Spermine / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*
D015342:DNA Probes; D007211:Indoles; D007364:Intercalating Agents; D008024:Ligands; D009841:Oligonucleotides; D003609:Dactinomycin; D013096:Spermine; C007293:DAPI; D001710:Biotin; D004247:DNA; D004996:Ethidium