spectrometric characterization of DNA binders
Many DNA binders interact with DNA via aromatic systems, or otherwise absorb in the range of UV/VIS light. This could cause background signals, which impair the FRET measurements. Therefore, we recorded spectra of the Atto dyes mixed with the different DNA binders (see Labbook: 11th August and here in Fig. 1).
Fig.1: Excitation and emission spectra of Atto 550 ddCTP resp. Atto 647N dUTP with and without different DNA binders
Atto dyes: 100 nM, DNA binders were added in a concentration according to 10x their KD (see references), i.e.: Hoechst 33258: 22 nM, DAPI: 18 nM, spermine: 48 µM, methyl green: 22 nM, ethidium bromide: 120 µM
EtBr without Atto 550 ddCTP refers to a spectrum with only 120 µM ethidium bromide, while Atto 550 ddCTP corrected for EtBr is the difference spectrum between ethidium bromide and EtBr without Atto 550 ddCTP
buffer: 0.5x TBE, 11 mM MgCl2; slit width: 5 nm; room temperature
Most DNA binders do not exhibit significant effects on the absorption and emission spectra of the Atto dyes and thus can be used for FRET measurements. Methyl green causes major effects and therefore will not be considered further for these experiments. Ethidium bromide absorbs and emits at similar wavelength as the both Atto dyes, but as the difference spectrum proves, the Atto dyes themselves are not affected. Consequently, ethidium bromide causes background signals which must be corrected, but otherwise can be used in our measurements.
We decided to concentrate on three DNA binding compounds, ethidium bromide as important example for an intercalator, DAPI as a minor groove binder and spermine as a major groove binder.