TITLE:
A Circular Dichroism Spectroscopy and Isothermal Titration Calorimetry Approach to Effectively Characterize the DNA Binding Mode of Compounds
AUTHORS:
Ruel Earl McKnight, Luke Timothy Marr, Gavin Smiley Gullickson, Kevin David Siegenthaler
KEYWORDS:
Isothermal Titration Calorimetry, Circular Dichroism Spectroscopy, DNA Binding Mode, Intercalation versus Minor Groove Binding, AT versus GC DNA
JOURNAL NAME:
Journal of Biophysical Chemistry,
Vol.17 No.4,
September
20,
2026
ABSTRACT: Isothermal titration calorimetry (ITC) and circular dichroism spectroscopy (CD) were used to investigate the binding of eight compounds [quinacrine, daunomycin, ethidium bromide, a naphthalene diimide, distamycin, 4’,6-diamidino-2-phenylindole (DAPI), berenil and Hoechst 33258] to two 20-bp DNA duplexes, [poly(dAdT)]2 and [poly(dGdC)]2. A primary goal was to illustrate the utility, ease and convenience of using ITC and CD in tandem to characterize the DNA binding mode of potential compounds, based on their preference for AT-rich versus GC-rich DNA sequences. Our CD studies showed that the four compounds (distamycin, DAPI, berenil, Hoechst 33258) known to bind DNA via the minor groove, elicited strong induced CD (ICD) signals between 300 - 400 nm when binding to the AT-rich DNA sequence. No ICD signal was observed for distamycin, DAPI and berenil when titrated into the GC-rich DNA sequence. Hoechst 33258, however, showed strong binding to both the AT-rich and GC-rich DNA sequences, indicating a mixed binding mode, likely involving DNA intercalation. All four of the compounds known to bind via intercalation displayed significant binding to both DNA sequences, with perturbations at the signature bands for DNA (~245 and 275 nm). Our ITC thermodynamic studies corroborated the CD results showing strong AT-DNA sequence binding for distamycin, DAPI and berenil (K~106-7 M−1) with only background binding (associated with the heats of dilution) to the GC-DNA sequence, while Hoechst 33258 showed binding to both DNA sequences, indicating an additional binding mode. ITC showed that the four intercalators bound strongly to both DNA sequences. Binding of the compounds to the DNA sequences were enthalpically driven with large negative enthalpy changes accompanied by generally small negative entropy changes.