Fig.1. Structures of the fluorescein nucleic acid dyes: TP3 and DAPI (5).

Fig.2. Schematic presentation of TP3 displacement from DNA helix followed by fluorescence quenching (5).

Methodology:Nucleic acid dye fluorescence quenching assays -­ A drug solution was added in 1 to 10uL aliquots to a solution of 20 uM sonicated calf thymus DNA and 2 uM dye in 3 ml of acetate buffer (pH 5.0). A fluorescence spectrum was recorded at each addition of the drug, and the intensity was noted at λem. The base level (buffer) was subtracted from each fluorescence measurement at λem. This value was then divided by the maximal fluorescence (dye and DNA only). The data were plotted against the concentration of each drug, and the C50 value of each was determined. C50 is the concentration of a drug at 50% fluorescence quenching of DNA-­bound dye. All fluorescence measurements were performed at 25˚C.

Confirmation of the fluorescence study results by NMR -­ Proton spectra were obtained on a JEOL ECX 300-­MHz spectrometer. Samples (800 uL) contained 0.5 mM imipramine, 0.5 mMpyrocatechol violet, or 0.3 mM janus green B and different amounts of sonicated calf thymus DNA in D2O. The spectra were recorded in 5-­mm NMR tubes.

Fig.3. Structures of the experimental molecules (5).

Results:Fluorescence quenching assays

Fig.5. Fluorescence quenching of DNA-­bound TP3 (s) and DAPI (d) by 11 different molecules (5).

Fig.7. NMR spectra of 0.5 mM pyrocatechol violet and pyrocatechol violet with equimolar amounts of DNA, both in D2O (5). In contrast to imipramine, the proton lines of the NMR spectra of pyrocatecholviolet did not shift on the addition of DNA, revealing a minor groove binding mode for pyrocatecholviolet.

Acknowledgments:Special thanks to Dr. Ekaterina Korobkova, Nikolay Azar, and Dr. Nathan Lents.Support for student stipends, supplies, and/or equipment used in this research was supplied by the Program for Research Initiatives for Science Majors (PRISM) at John Jay College. PRISM is funded by the Title V, HSI-­STEM and MSEIP programs within the U.S. Department of Education;; the PAESMEM program through the National Science Foundation;; and New York State’s Graduate Research and Teaching Initiative.

Determination of the drug–DNA binding modes using fluorescence-­based assays

Baibhav Rawal, Alicia K. Williams, Sofia Cheliout Dasilva, Ankit Bhatta,, Melinda Liu Ekaterina A. Korobkova *

Department of Science, John Jay College of Criminal Justice445 W 59th St., New York, NY 10019

Table 1. Probabilities of intercalative DNA binding mode and partition coefficients of 11 experimental molecules (5).Compound name Partition coefficient (log[P] I/G I%

NetropsinTartrazineAmaranthPyrocatechol violetBerenilNew coccineSunset yellow FCFImipramineBrilliant blue GCongo redJanus green

-­4.741-­1.766-­1.611-­1.533-­1.434-­0.425-­0.2651.0222.9713.8994.365

0.0200.0410.2700.0860.3100.0680.0321.1153.32.9

1.94.0217.9246.43.153947774

Note (5): The relative affinity, R, was presented as log[Kb]/C50. We hypothesized that a drug more effectively displaces a dye that has a similar DNA binding mechanism than a dye that has a different DNA binding mode. The ratio of the R coefficients (I/G) determined with TP3 and DAPI represents contributions of the two binding modes to the whole drug–DNA association mechanism. I/G = RTP3/RDAPI, where Rdye= log[Kb(dye)]/C50. C50 is the concentration of an experimental molecule at 50% fluorescence quenching of a bound dye. The units of C50 are mol/L (M). The percentage contribution of the intercalative mode (I%) was determined as I% = [1 + (I/G)-­1]-­1x100%.

Fig.4. Scatchard plots for nucleic acid dyes–DNA binding derived from fluorescence measurements (5). [DNA]/f is plotted versus (1 -­ f)-­1, where [DNA] is the concentration of the sonicated calf thymus DNA (in M per base pair) and f = (F(corr) –FD)/(Fmax(corr) – FD). (A) TP3–DNA Scatchard plot. The concentration of TP3 was 0.75 uM, and the concentration of DNA on the plot varied between 5.3 and 19 lM (bp). Inset: black line, fluorescence spectrum of TP3 alone;; red line, fluorescence spectrum of the solution containing 0.75 uM TP3 and 14 lM calf thymus DNA;; λex = 642 nm and λem = 661 nm. (B) DAPI–DNA Scatchard plot. The concentration of DAPI was 0.75 uM, and the range of DNA concentrations on the plot was 4.9 to 14 uM (bp). Inset: black line, fluorescence spectrum of DAPI alone;; red line, fluorescence spectrum of the solution containing 0.75 uM DAPI and 14 uM calf thymus DNA;; λex = 358 nm and λem = 461 nm. a.u., arbitrary units.

Fig.6 NMR spectra in the aromatic proton regions recorded from the solutions of imipramine and the different concentrations of DNA with different molar ratios of imipramine and DNA base pairs (5). As the concentration of DNA increased, the spectra became broader and the chemical shift changed by an increment ranging between -­0.5 and -­0.4 ppm. The upfield proton shift and the corresponding line’s broadening are proven to be a signature of an intercalative binding mode.

NMR -­ results shown for two of the three molecules studied

Estimating a relative affinity and a binding mode

Introduction:Therapeutic drugs (4) and environmental pollutants may exhibit high reactivity toward DNA bases and backbone. Understanding the mechanisms of drug-­DNA binding is crucial for predicting their potential genotoxicity. We developed a fluorescence analytical method for the determination of the preferential binding mode for drug-­DNA interactions. Two nucleic acid dyes were employed in the method: TO-­PRO-­3 iodide (TP3) and 40,6-­diamidino-­2-­phenylindole (DAPI). TP3 binds DNA by intercalation, whereas DAPI exhibits minor groove binding. Both dyes exhibit significant fluorescence magnification on binding to DNA (2, 3). We evaluated the DNA binding constant, Kb, for each dye (1). We performed fluorescence quenching experiments with 11 molecules and measured a C50 value for each compound. We determined preferential binding modes for these molecules. The values of the likelihood of DNA intercalation were correlated with the partition coefficients of the molecules. It was found that netropsin, berenil, pyrocatechol violet, sunset yellow, tartrazine, new coccine and amaranth bind preferentially to DNA by minor groove binding mechanism, while congo red, janus green and brilliant blue do so preferentially by intercalation. In addition, we performed nuclear magnetic resonance (NMR) studies of the interactions with DNA for the three molecules. The results were consistent with the fluorescence method described above. Thus, we conclude that the fluorescence method we developed provides a reliable determination of the likelihoods of the two different DNA binding modes.

References:1. Healy, E.F., Quantitative determination of DNA–ligand binding using fluorescence spectroscopy, J. Chem. Educ. 84 (2007) 1304–1307.2. Liu, Y., Danielsson, B., Fluorometric broad-­range screening of compounds with affinity for nucleic acids, Anal. Chem. 77 (2005) 2450–2454.3. Haugland, R.P., in: M.T.Z. Spence (Ed.), Handbook of Fluorescence Probes and Research Chemicals, Molecular Probes, Eugene, OR, 1996, p.153.4. Korobkova, E.A., Ng, W., Vethatratnam, A., Williams, A.K., Nizamova, M., Azar, N., In vitro studies of DNA damage caused by tricyclic antidepressants: a role of peroxidase in side effects of the drugs, Chem. Res. Toxicol. 23 (2010) 1497–1503. 5. Williams, A.K., Cheliout Da Silva, S., Bhatta, A., Rawal, B., Liu, M., Korobkova, E. A. Determination of the drug-­DNA binding modes using fluorescence-­based asays, Anal. Chem. 422 (2012) 66-­73.