SPECTROPHOTOMETRIC INVESTIGATION ON NUCLEIC
ACIDS INTERACTIONS WITH SOME ANTIVIRAL DRUGS
AND LIGAND MOLECULES:
Developing of New Methods for Nucleic Acids
Determination
Alaa A. Salem
Ibrahim M. Abdou
Islam K. Shams
CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
UAE UNIVERSITY
1
Background
Outlines
DNA Structure & Forms.
Interaction with drugs & small molecules : Binding Modes.
Experimental
Materials and Reagents.
Standard Solutions.
Methods.
Procedures
Spectral Results
Absorption Spectra.
Calibration Curves and Selectivity
Binding Curves
Analysis of synthetic and real samples
Fluorescence Spectra.
Optimization of Reaction Conditions.
Discussion of interaction mechanisms
Conclusions
2
Background
 DNA is the basic heredity material in
different cells.
It composed of Purines bases (A and G),
pyrimidines bases (T and C) attached to
sugar phosphate backbone.
 It is typically double stranded.
Its serves to transfer genetic material to
new cells.
3
DNA Forms
A-form
10 bases per turn
Right handed
Deep-narrow major
groove, broad shallow
minor groove
Hybrid DNA-RNA
B-form
10 bases per turn
Right handed
Wide major groove and narrow
minor groove with both moderate
depths
Most common form exist at
physiological pH (pH7)
Z-form
12 bases per turn
Left handed
Wide-shallow major
groove, deep-narrow minor
Stabilized at high salt
concentration
4
Interaction of Nucleic Acids with molecules: Binding modes
12 AO
Major
6 AO
Minor
Major and Minor grooves in DNA molecule (B-Form)
5
Interaction of Nucleic Acids with molecules: Binding modes
 Some chemical species such as water, metal ions, metal complexes,
small organic molecules, proteins and drugs are bind reversibly to
DNA.
 Interaction modes includes
A. Intercalation between the DNA base-pairs.
B. Groove-binding involves interactions or binding to edges of
base pairs through major or minor grooves of nucleic acids.
C. Electrostatic involve binding along the exterior helix.

Developing anti-cancer, anti-viral and anti-parasitic drugs is based
on their interruption of the DNA replication or transcription upon
binding.
6
+
+
Intercalation
Groove binding
Models of DNA interaction
Electrostatic
7
Interaction Modes of some Drugs
and dyes
Drug Name
Interaction Mode
ActinomycinD
Intercalation
Adolzelesin,
U-73,975
Minor groove
Ametantrone
Intercalation
Amiloride, Midamor
Intercalation
Bisantrene
Intercalation
Bizelesin,
U-77,779
Minor groove
CC-1065, U-56314
Minor groove
Chromomycin A3
Minor groove
CI-958, Parke-Davis
Intercalation
Daunomycin, Cerubidine
Intercalation,
directed into minor
groove
Drug Name
Mode of Interaction
DAPI
Minor groove/
intercalation
DACA
Intercalation
Distamycin A,
Stallimycin
Minor groove
Echinomycin
Bis-intercalation
Ethidium bromide
Intercalation
Hedamycin
Intercalation, threading
Hoechst 33258
Minor groove
mAMSA, amsacrine
Intercalation, threading
Mitomycin C
Minor/Major groove
Netropsin, T-1384
Minor groove
Nogalamycin
Intercalation, threading
o-AMSA
Intercalation
Tilorone
Intercalation
Tomamycin
Minor groove
8
C5
Motivations
5’
interaction modes, Searching for DNA Probes
Porphyrine Complexes
Z+
N
N
N
M
N
N
N
2+
3+
Bathophenanthroline Fe , Os ,
3+
Sm , Spectrochim Acta A,
2006, 65, 235-248
3’
T
G
C
C
C
T
C
A
A
A
T
A
C
A
A
C
A
A
A
A
C
A
A
C
A
G
A
A
A
C
A
A
A
A
C
A
A
A
A
A
C6
3’
5’
T
T
T
T
T
G
T
T
T
T
G
T
T
T
C
T
G
T
T
G
T
T
T
T
G
T
T
G
T
A
T
T
T
G
A
G
G
G
C
A
B11
A
T
T
T
C
C
A
A
G
A
A
A
C
G
C
A
A
G
G
T
A
A
T
T
G
G
G
G
A
A
A
A
A
T
G
A
G
A
C
A
5’
B12
T
G
T
C
T
C
A
T
T
T
T
T
C
C
C
C
A
A
T
T
A
C
C
T
T
G
C
G
T
T
T
C
T
T
G
G
A
A
A
T
D11 D12
A
T
T
T
C
C
A
A
G
A
A
A
C
G
C
A
A
G
G
T
A
C
C
T
T
G
C
G
T
T
T
C
T
T
G
G
A
A
A
T
Calf
Thymus
DNA
Human
9
DNA
Experimental
Standard solutions
phosphate buffer with different pHs
Reconstituted standard ss-DNA and standard ct-DNA
Standard drug and interfering solutions
Human DNA extracted from blood using QUIAGEN
recommended protocol.
10
Methods
 Different techniques have been used to study DNA
interaction
1.
2.
3.
4.
5.
UV-VIS photometry
Fluorimetry.
Atomic Force Microscopy (AFM).
Scanning Electrochemical Microscopy
Circular dichroism (CD) spectroscopy.
6.
7.
8.
9.
X-ray crystallography.
Nuclear Magnetic Resonance (NMR) spectroscopy.
Gel electrophoresis.
Electron microscopy.
11
Procedures
1. ssDNA Reconstitution
ssDNAs were
centrifuged at
7000 rpm for 2
minutes
2 ml of deionized
water (2 minutes) for
rehydration
Vortex for 15 seconds
Use directly or Keep in
fridge till use (up to 6
months)
2. Ct-DNA Reconstitution
10 mg Ct-DNA
1000 µg/ml ct-DNA
+
10 ml deionized water
+
2-3 EDTA Crystals
Overnight for DNA complete
dissolution at 0-4 oC
12
3. Quantitation of reconstituted Oligonucleotides
 An appropriate amount of re-suspended synthetic B11, B12, D11,
D12, C5 or C6, ct-DNA or human DNA was diluted, vortexed
and its absorbances were measured at 260 nm and 280 nm.
 The concentrations were calculated using the following formula
Concentration in
μg/ml
=
A260 x Weight per OD x dilution factor
• A260/A280 factor ≥ 1.8 for synthetic , natural ct-DNA, and human
DNA indicating high purity of extracts.
13
4. Hybridization of synthetic ss-Oligonucleotides
 Equi-molar amounts of the single stranded complementary
synthetic primers (D11 and D12 or C5 and C6) were mixed in
appropriate amount of phosphate buffer pH 7.
 The solution was vortexed, incubated at 50 oC and then left to
cool for 30 minutes at room temperature. Hybridized dsDNA
produced was used or kept in refrigerator till use.
5. Extraction of Human DNA
Done by QIAGEN protocol for extracting human DNA form blood sample.
20 µl
proteinase
K+200 µl
blood
200 µl
buffer AL
(vortexed
15 s & 10
min
incubate
at 56 oC
200 µl
absolute
ethanol,
vortexed
15s &
centrifuge
To QIAamp
Spin
Column,
centrifuged
at 8000 rpm
for min,
filtrate
discarded
500 µl AW1
buffer,
centrifuged
at 8000 rpm
for min,
filtrate
discarded
500 µl AW2
buffer,
centrifuged
at 14000
rpm for 3
min, filtrate
discarded
200 µl buffer
AE µl,
incubate at
25oC for
min,
centrifuge at
8000 rpm for
min
3-12 µg
of
Human
DNA
14
Spectrometric Measurements

Standard solutions of drugs or ABTS were prepared in
phosphate buffer pH 7.

Successive portions of diluted ssDNA, dsDNA, ct-DNA
and human DNA were added.

Absorbances or Fluorescence were recorded and corrected
for dilution.

Absorbence or fluorescence was plotted Vs nucleic acid
concentrations at max of DNA or interacting molecule.
15
Binding Sites and DNA – Drugs Association Constants
Were calculated using Rosenthal's graphical method
according to Rosenthal's method
0
16
Results
17
Absorption Spectra of 5-iodo-2-deoxyuridine
Band at 285
nm was
observed
1
100 μl portions of D11 (10 ppm) to 1 ml (10
ppm) 5-iodo
20
100 μl portions of D12 (10
ppm) to 1 ml (10 ppm) 5-iodo
20
1
1
20
100 μl portions of ds(D11-D12) (10 ppm) to 1 ml
(10 ppm) 5-iodo
18
Absorption Spectra of ABTS
Bands at 260,
and 340 nm
were
observed
1
24
100 μl portions of C5 (20 ppm) to 1 ml (20
ppm) ABTS
1
24
100 μl portions of ds(C5-C6) (20 ppm) to 1
ml (20 ppm) ABTS
19
Optimization of reaction conditions
1. Reaction time and stability
Completed in 1-2 minutes at room temperature and remain
constant for 24 hours.
2. Addition order of reagents
No significant change (DNA, drug, buffer).
20
Calibration Curves (Levofloxacin)
Change in
Absorbance
at 290 nm
D11 + levofloxacin
D12 + levofloxacin
ds(D11,D12) + levofloxacin
ct-DNA + levofloxacin
0.7
0.6
Abs. at 290 nm
0.5
0.4
0.3
0.2
0.1
0
1
2
3
4
5
6
7
levofloxacin Conc. in ug/ml
D11, D12, ds(D11,D12), ct-DNA + levofloxacin (10ppm, pH7, at 290 nm)
21
Reported values for calibration graphs at 260 and 290 nm.
Nucleic acid
Linear ranges
Linear regression
(g/ml)
slope
R2
Using levofloxacin as probing species (290 nm)
D11 (ssDNA)
0.9-6.6
Y=0.1X+0.0375
0.1
D12 (ssDNA)
0.9-6.6
Y=0.1X+0.0549
0.1
dsDNA
0.9-6.4
Y=0.1X+0.0168
0.1
ct-DNA
0.9-6.6
Y=0.1X+0.0522
0.1
0.99
0.99
0.99
0.99
22
Reported values for calibration graphs at 260 and 475 nm.
Using , ABTS as probing species (260 nm)
C5 (ssDNA)
0.9-6.6
Y=0.01X+0.1229
0.01
C6 (ssDNA)
0.9-6.6
Y=0.01X+0.1558
0.01
dsDNA
0.9-6.6
Y=0.01X+0.153
0.01
ct-DNA
1.6-6.6
Y=0.01X+0.1017
0.01
0.97
0.99
0.99
0.99
Using , ABTS as probing species (340 nm)
C5 (ssDNA)
0.9-6.6
Y=-0.03X+0.3477
0.03
C6 (ssDNA)
0.9-6.6
Y=-0.04X+0.4565
0.04
dsDNA
0.9-6.6
Y=-0.04X+0.4506
0.04
ct-DNA
0.9-6.4
Y=0.03X+0.316
0.03
0.98
0.99
0.99
0.98
23
Selectivity
Effects
of
some
interfering substances on
ABTS–ct-DNA
intercalates, buffer pH 7.
A 10-7 M interfering
solutions were used
Interfering
Agents
Conc.
Mole/L
Alanine
A%
225 nm
255 nm
340 nm
6.67 x 10-7
-13.06
-11.39
-12.5
CaCl2
6.67 x 10-7
2.99
1.6
-12.8
CdCl2
6.67 x 10-7
-9.96
-8
-8.59
CoCl2
6.67 x 10-7
-11.08
-9.54
-9.09
CuSO4
6.67 x 10-7
19.14
30.07
10.0
FeSO4
6.67 x 10-7
14.50
14.93
2.44
Glycine
6.67 x 10-7
-9.45
-9.27
-10.32
HgCl2
6.67 x 10-7
12.60
-19.45
-8.94
D-L-Valine
6.67 x 10-7
-7.05
-2.68
-6.20
MnSO4
6.67 x 10-7
5.17
-6.71
-9.56
Pb(NO3)2
6.67 x 10-7
4.54
11.01
-5.6
Zn(Cl)2
6.67 x 10-7
-8.92
-8.97
-9.85
 A% values between 2.4-12.5%, 1.6-14.45% and 2.99-19.14% were obtained at 340,
255 and 225 nm, respectively.
 Since interfering species can be eliminated during DNA purification, a good selectivity
24
exists for the interaction between ABTS and ct-DNA at 340 nm.
Binding Sites and Association Constants

Based on Rosenthal’s graphical model
Binding curve of C6 Vs AzBTS (20ppm, pH7, 340 nm)
2.75
y = -216051x + 0.9593
2
R = 0.991
Ao/A
2.15
1.55
0.95
-8.E-06 -7.E-06 -6.E-06 -5.E-06 -4.E-06 -3.E-06 -2.E-06 -1.E-06 0.E+00
-(1-A/Ao) * C
Binding curve for
synthetic ssDNA
with ABTS at 340
nm .
Binding curves' slopes (K), intercepts, correlation coefficients (R2), number of
binding sites (n) and linear equations at 260 and 340 nm based on interaction
between DNA and ABTS
Nucleic
acid
λmax
Conc.
K Assoc.
Const.
intercept
R2
Equation
C5
260
1.22E-05
4.2 x 104
0.897
0.90
Y= -41610X + 0.8975
C5
340
1.22E-05
2 .1x 104
1.004
0.99
Y= -21288X + 1.0041
C6
260
1.22E-05
4 .6x 104
0.933
0.96
Y= -46207X + 0.9332
C6
340
1.22E-05
2 .2x 104
0.959
0.99
Y= -21605X + 0.9593
ds (C5,C6)
260
1.22E-05
4 .4x 104
0.927
0.95
Y= -44341X + 0.9278
ds (C5,C6)
340
1.22E-05
2.2 x 104
0.956
0.99
Y= -21946X + 0.9565
ct-DNA
260
1.22E-05
3.4 x 104
0.845
0.91
Y= -33749X+ 0.8459
ct-DNA
340
1.22E-05
2.2 x 104
0.928
0.99
Y= -22262X + 0.9288
Average number of binding constant 4.0
26
Analysis of Synthetic and Real Samples
Determination of ct-DNA and human DNA based on the 260, 285, 290, 340
nm bands using the calibration curves from synthetic dsDNA.
Concentration
Found (g/ml)a
(g/ml)
a) Using 5-iodo-2’-deoxyuridine as probing species
Synthetic dsDNA
3.00
3.20
Recovery%
R.S.D
106.60
3.33%
5.00
5.10
102.00
2%
3.00
2.95
98.30
2.67%
2.80
93.33
3.33%
3.10
103.33
3.33%
2.95
98.30
3.33%
2.90
96.67
3.33%
310
103.33
3.33%
5.00
4.90
98.00
2%
3.00
3.10
103.33
3.33%
2.90
96.67
3.33%
2.90
96.67
3.33%
5.00
5.10
102.00
2%
Ct-DNA
3.00
3.10
103.33
3.33%
Human DNA
3.00
2.80
93.33
3.33%
Nucleic acid
Ct-DNA
Human DNA
3.00
b) Using levofloxacin as probing species
Synthetic dsDNA
3.00
Ct-DNA
3.00
Human DNA
3.00
c) Using ABTS as probing species
Synthetic dsDNA
3.00
Ct-DNA
Human DNA
3.00
d) Using as rifampicin as probing species
Synthetic dsDNA
3.00
27
Analysis of Synthetic and Real Samples
 DNA in extracts from calf thymus and human blood were
determined using the calibration graph for synthetic DNA .
 Recoveries%
of 93.33-106.60 were obtained for natural
calf thymus and human DNA using 5-iodo-2’-deoxyuridine,
levofloxacin, ABTS and rifampicin.
 Standard deviations in the range of (2-3.33 %).
 This reveals clearly that the proposed methods are reliable,
simple, and practicable.
28
Fluorescence spectra of ABTS
 Reduced ABTS show intense fluorescence bands around
465, 680, and 780 nm using 230, 340 and 390 nm as
excitation wavelengths, respectively.
Addition of C5, C6, ds(C5-C6), ct-DNA or human DNA
decreased the fluorescence intensities due to the intercalation
of nucleic acids with ABTS
29
Intensity (a.u.)
1000
10 μl portions of C5 (20 μg/ml)
to 1.0 ml of ABTS (20 μg/ml)
in phosphate buffer pH 7.
(I)
1
(III)
(II)
800
600
7
400
200
0
500
600
700
Wavelength (nm)
800
(III)
1000
Intensity (a.u.)
10 μl portions of human DNA
(20 μg/ml) to 1.0 ml of ABTS
(20 μg/ml) in phosphate buffer
pH 7.
(I)
1
(II)
800
600
7
400
200
0
500
600
700
Wavelength (nm)
800
30
Calibration curve of C5, C6, ds(C5,C6), and ct-DNA using AzBTS (20 ppm, pH 7, 465 nm)
Calibration curves for ssDNA
and dsDNA at 465, 680 and 780
nm using 20.0 ppm ABTS.
1200
C5 + AzBTS
465 nm.
1000
C6 + AzBTS
600
ds(C6,C5) +
AzBTS
Dynamic ranges between 0.2-1.1
µg/ml were obtained.
400
200
0
0.2
0.4
0.6
0.8
1
1.2
Nucleic acid
Conc. ug/ml
Calibration
curve of C5, C6, ds(C5,C6), and ct-DNA using AzBTS (20 ppm, pH 7, 680 nm)
800
C5 + AzBTS
680 nm
700
600
C6 + AzBTS
500
ds(C6,C5) +
AzBTS
400
ct-DNA +
AzBTS
300
200
Calibration curve of C5, C6, ds(C5,C6), and ct-DNA using AzBTS (20 ppm, pH 7, 780 nm)
100
1000
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
C5 + AzBTS
900
780 nm
800
Nucliec acid Conc. ug/ml
C6 + AzBTS
700
Int. at 780nm
0
Int. at 680nm
Int. at 465nm
800
ds(C6,C5) +
AzBTS
600
500
ct-DNA +
AzBTS
400
300
200
100
31
0
0
0.2
0.4
0.6
0.8
Nucleic acid Conc. ug/ml
1
1.2
1.4
Selectivity on ct-DNA-ABTS intercalate
Effect of some interfering substances showed average F% changes
up to 24.07% at the 465, 680 and 780 nm bands.
Since investigated interferants can be eliminated during DNA
purification, a good selectivity could be assumed for the interaction
between ABTS and ct-DNA.
Effects of some interfering solutions on ABTS–ct-DNA intercalate in phosphate
buffer pH 7. A 10-7 M interfering solutions were used.
Interfering
substance
Conc.
mol/l
Alanine
CaCl2
CdCl2
CoCl2
CuSO4
FeSO4
Glycine
HgCl4
D-L-Valine
MnSO4
Pb(NO3)2
ZnCl2
F%
465 nm
680 nm
780 nm
6.67E-07
6.67E-07
-4.93
10.1
19.45
6.60
-13.72
0.00
6.67E-07
6.67E-07
-1.79
6.12
7.13
-1.37
0.01
2.03
6.67E-07
6.67E-07
high
high
high
9.71
high
high
6.67E-07
6.67E-07
21.67
-17.10
-10.25
-6.83
-2.25
-0.43
6.67E-07
6.67E-07
29.07
-5.70
-4.73
11.29
-24.34
-5.95
6.67E-07
6.67E-07
115.19
-72.31
-47.79
4.84
-13.60
-6.22
Association constant and Binding Sites
Association constants in the ranges of:
9.0x105-3.0x106 (ssDNA-ABTS) at 465, 680, and 780 nm.
8.0x105-3.0x106 for dsDNA-ABTS at 465, 680, and 780 nm.
6.0x105-5.0x106 for ct-DNA-ABTS at 465, 680, and 780 nm.
Average Binding sites using 2.3910-6 M DNA were in the
range of 3-4 for ct-DNA
Such variations could be attributed to changes in binding
curves' slopes and intercepts as well as to the complexity of
DNA molecules.
34
Analysis of Synthetic and Real Samples
Concentration
(g/ml)
Found (g/ml)
Recovery
%
R.S.D
ssDNA (C5)
1.00
1.10
110.0
10.00
ssDNA (C6)
1.00
0.95
95.00
5.00
dsDNA (C5C6))
1.00
1.05
105.00
5.00
Ct-DNA
1.00
0.96
96.00
10.00
ssDNA (C5)
1.00
1.00
100.00
8.50
ssDNA (C6)
1.00
1.05
105.00
7.40
dsDNA (C5C6))
1.00
0.95
95.00
9.00
Ct-DNA
1.00
0.93
93.00
12.00
ssDNA (C5)
1.00
1.00
100.00
9.00
ssDNA (C6)
1.00
0.98
98.00
6.02
dsDNA (C5C5))
1.00
1.05
105.00
5.00
Ct-DNA
1.00
0.96
96.00
10.00
Nucleic acid
At 465 nm
Determination
results based
on the 465,
680 and 780
nm using
ABTS.
At 680 nm
At 780 nm
35
Analysis of Synthetic and Real Samples
 DNA in extracts from calf thymus and human blood were
determined using the calibration graph for synthetic DNA .
 Recoveries% of 93.00-110.00 were respectively obtained
for natural calf thymus and human DNA using ABTS.
 These recoveries reflected relative standard deviations in
the range of (5-12 %).
 This reveals clearly that the proposed methods are reliable,
simple, and practicable.
36
Mode of Interaction
 Structure planarity, non-negative charges and quenching of
absorption intensity are the vital evidences required for an efficient
intercalative mechanism.
 5-iodo-2’-deoxyuridine,
levofloxacin and rifampicin have proven
these features with different extents. Quenching of absorption intensity
at their max is an evidence of intercalating these drugs into the stacking
base pairs of nucleic acids. Thus, binding of these drugs with DNA
can be assumed to occur via an intercalation mode.
 ABTS in its reduced form is negatively charged
compound ????. Therefore, mixed groove binding and
intercalation mechanism might be suggested for its
interaction with DNA.
37
Conclusion
1. Interaction of synthetic ssDNA, dsDNA and ct-DNA with 5-iodo-2’-deoxyuridine,
levofloxacin, ABTS,
Fluorimetrically.
and
rifampicin
were
studied
photometrically
and
2. An intercalation interaction mode was proposed for the binding of ssDNA, dsDNA,
and ct-DNA with 5-iodo-2’-deoxyuridine, levofloxacin and rifampicin drugs.
3. A mixed groove binding – intercalation mode might be proposed for the interaction
between DNA and ABTS.
4. Reaction of ABTS with ct-DNA has shown less interference towards some cations
and amino acids that were tested and satisfactory results were obtained.
5. Spectrophotometric and spectrofluorimetric methods for determining synthetic and
natural
DNA were developed based on intercalating DNA with 5-iodo-2’deoxyuridine, levofloxacin, ABTS, and rifampicin.
6. Developed methods have shown good sensitivity, rapidity and reproducibility when
applied for determining nucleic acids in synthetic and biochemical samples.
Acknowledgment
UAEU financial support
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