BIOPHYSICAL METHODS

DNA-ETHIDIUM BROMIDE INTERACTION

NAME
ROLL NO.
THEORY :
Ethidium bromide [3,8-diamino-5-ethyl-6-phenylphenanthiridinium
bromide]is commonly used to probe DNA structure in drug-DNA and
protein-DNA interactions. It binds nucleic acids via intercalative mode

and cause major changes to DNA and RNA structures. As a drug, it has

trypanocidal, antibacterial and anti viral activities. It has mutagenic as well

as carcinogenic activity as it inhibits DNA.

Ethidium Bromide contains tricyclic phenanthridine ring system that is

able to interact with stacked base pairs of double stranded DNA. EtBr
binds DNA with no apparent sequence preference once every 45 base
pairs Ethidium is capable of forming close van der Walls contacts with the
base pairs and due to that it can bind to the hydrophobic interior of the
DNA molecule.
Two main modes of binding to native DNA have been suggested. The
primary and generally stronger mode of binding has been interpreted as
intercalation where a part of the ethidium ion sandwiches between
adjacent base pairs. It is also shown to be anti co-operative binding
preferably at smaller Ethidium bromide to DNA concentration ratio. The
second and generally weaker mode of binding is interpreted as Non
intercalative binding which is thought to be an electrostatic interaction
between the phosphate groups in the double-stranded nucleic acid
backbone and the dye molecules. It shows shows positively co-operative
binding, most evident at low salt and high Ethidium Bromide
concentration.
The in vitro study of EtBr-DNA binding can be monitored by analysing
the photophysical changes of Ethidium upon DNA addition. Ethidium

binds to DNA duplexes with moderate affinity (Kd ranges from 0.2
200M) and variable stoichiometry (3 6 equivalents of Ethidium per
helical repeat) depending on the sequence of the duplex. Ethidium binds
to different duplex nucleic acids with a free energy range of 5.38.8
kcal/mole, depending on the structure and sequence of the nucleic acid.
The unwinding of DNA from the binding of intercalating drugs relaxes
negative supercoils. The unwinding angles of EtBr are about 26 per
intercalated molecule respectively, it takes ~13 intercalated molecules to
unwind the helix by 360 or remove one turn of the DNA double helix
(with an unwinding angle of 26). Unwinding from intercalation lowers
the value of LO. Therefore, as an increasing number of molecules
intercalates into DNA, the value of LO decreases. In case of circular DNA,
which has got no free ends, after enough EtBr has bound to DNA it
causes circular DNA to become twisted to form a 8 shaped figure. As
more Ethidium binds to DNA, the figure gets more twisted, until a time
comes when it is unable to twist any further.
A time comes when the DNA gets saturated with EtBr, till there is no
further increase in kinking of DNA.
Non intercalative binding, relatively weak form of binding, that only
occurs when the space between the base pairs of DNA have become
saturated with EtBr. The former (intercalative) mode of binding is stronger
one, while the latter is weak. In
spectrophotometric titration of EtBr binding to DNA, an Isosbestic point is
observed at 518nm. Free Ethidium Bromide has a maximum absorbance
at 485.9761nm. As more and more EtBr binds to DNA, the free Ethidium
bromide amount decreases. Therefore, the absorption (maximum)
decreases accordingly with subsequent addition of DNA, with a spectral
shift in maximum absorbance.
PROTOCOL:
1mg EtBr was weighed in a digital weighing balance. It was mixed
thoroughly with 1ml distilled water.
700uL distilled water was taken in a quartz cuvette and through the
spectrophotometer, base line correction was meted out to cancel
out the noise of several peaks due to air as well as the solvent itself.
To it, 20uL of EtBr was added, mixed by inversion by using paraffin
paper on top of the cuvette opening.
Since native EtBr peak was small, 20uL was further added to the
cuvette containing 720uL solution to attain standardisation.
10uL EtBr was again added till threshold is reached and peak gets
flattened. The wavelength maxima of the Absorbance spectrum was
noted and the corresponding absorbance value was taken for
further calculation.
3. 10l of stock DNA solution was added to the above solution in a
cuvette and its absorbance was noted.
The above step was repeated several times with subsequent
addition of DNA (15,20,25,35,45,55,75,85,95,105 l) and the
absorbance was noted until a time, when there was no significant
spectral shift was noticed.

DNA SOLUTION: Calf thymus DNA was dissolved in distilled water to

attain a concentration of 2mg/ml(20l DNA solution taken in an
eppendorf, to which 980l of distilled water added). Absorbance of this
solution was measured at 260nm (0D260).
From the absorbance, concentration of DNA stock solution was
calculated using Lambert Beers Law.

OD = cl

Where OD = Optical density (Absorbance)

= Molar extinction coefficient (for DNA = 6600l/mol/cm)

C = Concentration of DNA

l = Path length through which light travels

From this, the concentration of DNA in each addition was found out.

PLOT :
1. A graph depicting the differences in absorbances in various
concentrations of DNA-EtBr was plotted; with Optical
Density(Absorbance) in Y axis and Wavelength in X axis. The
isobestic point was determined from the plot.
2. A graph was plotted with A/A in Y axis, where A = A-Ax and
where, A = maximum absorbance (at the wavelength where the
peak was observed).
From this graph, binding affinity (K) could be found out. K is the
dissociation constant indicating how strongly the ligand is bound to
DNA and K~1/C .
3. A hill plot for DNA-EtBr interaction was plotted. The hill plot depicts
two straight lines indicating two different modes of binding.
Here, ln/1- is plotted in Y axis with ln[DNA] in X axis; where
= fraction of binding sites of DNA filled with Ethidium Bromide.
 Mathematically, is obtained by following the formula
= (O.D.EtBr O.D.EtBr-DNA)/(O.D.EtBr O.D.min)

INSTRUMENTATION :
The experiment was carried out in a spectrophotometer. The
instrument consists of a light source, a monochromator (for
wavelength selection), a transparent sample holder called a
cuvette, a light detector and a meter or recorder, for measuring
the output of detector. In our instrument the readings from
detector were displayed in an inbuilt monitor, along with a graph
and the same data and graph were printed on printer paper
associated with the spectrophotometer.
We made a measurement of the light transmitted by solvent
(here water), then the instrument is adjusted to read zero
absorbance when the solvent alone is measured(zeroing the
instrument). Then, the instrument so adjusted the absorbance of
sample is read directly.
In our experiment, as EtBr is red in colour, so we set the
instrument to scan and record absorbance value(optical density)
over arrange of 400-600nm.
We obtained the peak absorbance for Ethidium Bromide at
489.9289 nm.

CALCULATION :
For DNA Concentration :
 OD of DNA at 260 nm (OD 260) = 0.2707 = A

A = Cl ; = 6600 L/Mole/cm

0.2707 = Cl

C = 0.2707 / 6600 moles/L

= 4.1015 x 10-5 moles/L

= 41.015 x 10-6 moles/L

= 41.015moles/L

1 L solution contains = 41.015moles

106 l solution contains = 41.015moles

1l solution contains = 41.015 x 10-6 moles

10l addition of this DNA solution corresponds to addition of 41.015 x

10-5moles of DNA

10l addition corresponds to 4.1015 x 10-4 moles of DNA

15l addition corresponds to 6.15225 x 10-4 moles of DNA

So, the concentration of DNA in the solution during successive additions

was calculated and recorded in Table 1.

 TABLE 1:

No. of (nm) A AX A=(A-AX) A/A [DNA]x10-4

Observations (Y-axis
value)
1. (Only 489.928 0.191136 0.191136 0 0 0
EtBr) 9
DNA
(0l)
2. DNA(10 0.191136 0.190046 0.00109 0.005703 4.1015
l)
3. DNA (15 0.191136 0.18411 0.007026 0.036759 6.15225
l)
4. DNA (20 0.191136 0.174994 0.016142 0.084453 8.203
l)
5. DNA (25 0.191136 0.171951 0.019185 0.100374 10.25375
l)
6. DNA (35 0.191136 0.168719 0.022417 0.117283 14.35525
l)
7. DNA (45 0.191136 0.162697 0.028439 0.148789 18.45675
l)
8. DNA (55 0.191136 0.153427 0.037709 0.197289 22.55825
l)
9. DNA (75 0.191136 0.148242 0.042894 0.224416 30.76125
l)
10. DNA (85 0.191136 0.144136 0.047 0.245898 34.86275
l)
11. DNA (95 0.191136 0.134713 0.056423 0.25362 38.96425
l)
12. DNA 0.191136 0.139597 0.051539 0.269646 43.06525
(105 l)
PLOT OFA/A VERSUS [DNA]:

0.35

0.30

0.25 100% saturation

0.2425

0.20
A/ A K= 1/ [DNA] (M )
-1

0.15 -4
0.12125 = 1/ [12.8355*10 ]
-1
= 779.90 M
0.10

0.05

12.8355
0.00
0 20 40

[DNA] * 10-4
 No. of (nm) A AX A=(A- W 1- /1- Ln(/1-) Ln[DNA]
Observations AX) Y-axis X-axis

1. (OnlyEtBr) 0.19474 0 0 1.000 0 value value-

(DNA0l)

2. DNA 0.19474 0.19481 -0.00007 -0.0012 1.0012 -0.00119 - 3.7139

(10L) 3

3. DNA(15l) 0.19474 0.18756 0.0072 0.1218 0.8782 0.1387 -0.1.9755 4.1194

2
4. DNA(20l) 485.9761 0.19474 0.17499 0.0168 0.0591 0.284 0.716 0.3966 -0.9247 4.4071
4
5. DNA(25l) 0.19474 0.1736 0.0211 0.357 0.643 0.5552 -0.5884 4.6302

6. DNA(35l) 0.19474 0.1690 0.0257 0.04349 0.5651 0.7696 -0.2619 4.9667

7. DNA(45l) 0.19474 0.16138 0.0334 0.5651 0.4349 1.2994 0.2619 5.2180

9
8. DNA(55l) 0.19474 0.1525 0.0422 0.714 0.286 2.4965 0.9149 5.4187

9. DNA(75l) 0.19474 0.14575 0.0489 0.8274 0.1726 4.7937 1.5673 5.7288

6

10. DNA(85l) 0.19474 0.14117 0.0536 0.9069 0.0931 9.7411 2.2764 5.8540

11. DNA(95l) 0.19474 0.1388 0.05591 0.9459 0.0541 17.4843 2.8613 5.9652

12. DNA(105l 0.19474 0.1356 0.0591 1.000 0 1 0 6.0653

)
A= ODEtBr

AX= OD(EtBr-DNA)

A=(A- AX)= ODEtBr- OD(EtBr-DNA)

W= ODEtBr-ODMIN

= ODEtBr- OD(EtBr-DNA)/W

PLOT OF ln(/1-) versus ln[DNA]:

3 y = 3.278x - 9.355
2

0
ln(/1-)

0 1 2 3 4
-1 ln(/1-)
ln (/1-)
-2

-3

-4
y = 1.9677x - 5.322
-5

-6
ln [DNA]
INTERACTION OF DNA WITH ETHIDIUM BROMIDE

INTERPRETATION :

Ethidium Bromide showed a maximum absorbance at 485.9761 nm.

An isobestic point was obtained at 518nm.
On addition of DNA, more and more EtBr gets stacked in between
the base pairs, thereby the amount of free EtBr in solution was
generally reducing, which had an effect on the maximum
absorbance value. The OD peak value decreased with subsequent
 addition of DNA to Ethidium Bromide. This was indicated in the
absorbance versus wavelength plot.
A marked red shift of the peak values towards higher wavelength
was obtained, with more and more addition of DNA. As EtBr binds
to DNA, it distorts the duplex structure producing kinks in DNA. It
also causes unwinding of DNA and may introduce superhelicity.
There is a possibility, that the overall tertiary structure of DNA gets
more affected than the secondary structure. Since EtBr binds or
intercalates between the bases, it causes the lengthening and
unwinding of DNA. The distance between the base pair increases to
such an extent that it always equals the distance between sugar
phosphate linkage. In this case, a very little rotation is possible
about the helix, as it tends to unwind. Due to such unwinding of
DNA, bases are more exposed to polar aqueous environment,
which is the cause of red shift.
The plot obtained from A/A versus [DNA], gives the binding
affinity of EtBr and DNA in terms of K. K value comes to about M-1
(K=affinity constant).
The first mode of binding is a strong non co-operative, intercalative
type, where EtBr gets stacked in between the bases. This type of
binding is preferred at smaller EtBr to DNA concentration. This type
of binding usually gets saturated after enough EtBr has bound to
DNA, such that there are no more intercalation possible.
The second mode of binding is co-operative and non-intercalative
type, which only occurs after intercalation binding has reached a
saturation point.
 The quantitative estimation could be done by obtaining slopes of
these two lines.

n1 = 3.278 n2 = 1.9677

SIGNIFICANCE :
Binding affinity of one ligand with DNA shows how tightly the
ligand is bound to DNA; indicating the amount of affinity of ligand
to DNA. This maybe compared with binding affinity of another
ligand and can be put to use in drug designing, as to find which is
more potent.
EtBr can be used for site directed mutagenesis.
The interaction between DNA-intercalating agents provides
information regarding the biological activity of planar
chromophore.