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A surface plasmon resonance method for detecting multiple modes of DNA-ligand interactions
M L Ciolkowski1, M M Fang, M E Lund
1Pharmaceutical Development, Pharmacia and Upjohn Inc, Kalamazoo. MI 49007-4940, USA. mary.l.ciolkowski@am.pnu.com
Journal of Pharmaceutical and Biomedical Analysis
|June 17, 2000
Summary
A new surface plasmon resonance (SPR) method accurately quantifies low molecular weight compound binding to DNA. This technique is effective for studying intercalation, minor groove binding, and electrostatic interactions, with results aligning with literature values.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Studying molecular interactions between small compounds and DNA is crucial for drug discovery and understanding biological processes.
- Accurate quantification of binding affinities (Keq) is essential for characterizing these interactions.
- Existing methods may have limitations in sensitivity or applicability to various binding modes.
Purpose of the Study:
- To develop and validate a simple, general surface plasmon resonance (SPR) method for detecting and quantifying the binding of low molecular weight compounds (200-1,200 Da) to double-stranded DNA.
- To investigate different binding modes, including intercalation, minor groove binding, and electrostatic interactions.
- To assess the influence of co-solvents like DMSO on DNA-compound interactions.
Main Methods:
- Utilized surface plasmon resonance (SPR) spectroscopy to measure binding responses on a DNA-modified surface.
- Employed various known DNA-binding compounds: Ethidium bromide (intercalation), Actinomycin (intercalation/minor groove), DAPI (minor groove), and Spermine (electrostatic).
- Determined binding affinity (Keq) by analyzing steady-state SPR responses and fitting binding isotherms. Tested effects of DMSO co-solvent.
Main Results:
- The SPR method successfully detected and quantified binding for compounds representing different interaction modes.
- Measured Keq values for Ethidium bromide (1.8 x 10^5 M^-1), Actinomycin (1.9 x 10^6 M^-1), DAPI (1.8 x 10^6 M^-1), and Spermine (1.7 x 10^4 M^-1).
- No significant effect on Keq was observed for Ethidium bromide binding in the presence of 0-5% DMSO, indicating method robustness.
Conclusions:
- The developed SPR method provides a simple and general approach for quantitating low molecular weight compound binding to DNA.
- The technique accurately characterizes diverse DNA binding interactions, including intercalation, minor groove binding, and electrostatic interactions.
- Results are in excellent agreement with literature values, validating the SPR method's reliability for biophysical studies.