1. 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 mM
pyrocatechol 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 pyrocatechol
violet did not shift on the addition of DNA, revealing
a minor groove binding mode for pyrocatechol
violet.
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 Justice
445 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%
Netropsin
Tartrazine
Amaranth
Pyrocatechol violet
Berenil
New coccine
Sunset yellow FCF
Imipramine
Brilliant blue G
Congo red
Janus green
-4.741
-1.766
-1.611
-1.533
-1.434
-0.425
-0.265
1.022
2.971
3.899
4.365
0.020
0.041
0.270
0.086
0.310
0.068
0.032
1.1
15
3.3
2.9
1.9
4.0
21
7.9
24
6.4
3.1
53
94
77
74
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.
![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 mM
pyrocatechol 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 pyrocatechol
violet did not shift on the addition of DNA, revealing
a minor groove binding mode for pyrocatechol
violet.
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 Justice
445 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%
Netropsin
Tartrazine
Amaranth
Pyrocatechol violet
Berenil
New coccine
Sunset yellow FCF
Imipramine
Brilliant blue G
Congo red
Janus green
-4.741
-1.766
-1.611
-1.533
-1.434
-0.425
-0.265
1.022
2.971
3.899
4.365
0.020
0.041
0.270
0.086
0.310
0.068
0.032
1.1
15
3.3
2.9
1.9
4.0
21
7.9
24
6.4
3.1
53
94
77
74
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.](https://image.slidesharecdn.com/2e251b1f-408b-4209-8ba4-850f0b5baa02-170131170303/85/prism-spring-2012-drug-dna-binding-poster-1-320.jpg)
