The interactions of bis-phenanthridinium – adenine conjugates
with polynucleotides
Lidija-Marija Tumir, Filip Šupljika, Ivo Piantanida
1
2
3
Scheme S1. Conformations of studied compounds obtained by MD simulations. (1)
Table S1. Groove widths and depths for selected nucleic acid conformations.(2,3)
Groove width [Å]
Groove depth [Å]
major
minor
major
minor
a
poly dAdT – poly dAdT
11.2
6.3
8.5
7.5
b
poly dA – poly dT
11.4
3.3
7.5
7.9
c
poly rA- poly rU
3.8
10.9
13.5
2.8
a
poly dGdC – poly dGdC
13.5
9.5
10.0
7.2
a
B-helical structure (e.g. B-DNA); b C-helical structure (e.g. C-DNA). c A-helical structure
(e.g. A-DNA).
Table S2. Stability constants (logKs)a for complexes of 3 (4) (citrate buffer, pH = 5.0, c = 0.03
mol dm-3) at pH 5 with ss- and ds-polynucleotides calculated by processing of fluorimetric
titrations by means of Scatchard equation (5)
3
poly dA – poly dT
5.4
poly dAdT – poly dAdT
6.2
poly dG – poly dC
6.2
poly dGdC – poly dGdC
b
poly rA–poly rU
5.3
poly
rAH+
– poly
rAH+
5.6
poly U
b
a
Values of log Ks are recalculated according Scatchard equation for fixed n=0.1 for easier comparison; coefficients of
correlation were >0.989-0.999 for all calculated Ks; processing of titration data by means of Scatchard equation gave values of
ratio n=0.10.05 for most complexes.
bThe emission changes at excess of polynucleotide were too small or linear for accurate calculation of binding constants.
Thermal melting studies
Table S3 Tm-valuesa,b (°C) of different ds-polynucleotides with 1-2 at different ratios rc; pH = 5.0
(I=0.03 mol dm-3, sodium citrate buffer).
1
2
rc =
0.1
0.2
0.3
0.1
0.2
0.3
poly dAdT - poly dAdT
5.7
5.5
5.5
5.4
5.5
5.9
poly A - poly U
d
-1.2 / 0
d
-1.0 / 0
d
-1.5 / 0
d
0.9 / 0
d
0.9 / 1.2
d
1.5 / 0
poly dA - poly dT
1.8
2.4
3.5
0.8
1.1
0.8
poly AH+ – poly AH+
1.1
2.3
4.6
0.6
0.9
1.0
a
Error in Tm :  0.5°C;
b
values were corrected for absorbance of examined compound at different ratios r
c
r = [compound] / [polynucleotide];
d
biphasic transitions: the first transition at Tm = 28.5 oC was attributed to denaturation of polyA-polyU
and the second transition at Tm = 80.1 oC was attributed to denaturation of poly AH+-poly AH+ since
poly A at pH=5 is mostly protonated and forms ds-polynucleotide. (3)
Isothermal titration calorimetry (ITC):
Isothermal titration calorimetry (ITC) is used to characterize the thermodynamics of
interactions of small molecules with macromolecules (DNA, RNA), providing valuable
information about the enthalpic and entropic contributions to the binding Gibbs energy. The
data points in figures S1 and S2 reflect the experimental injection heats while the solid lines
reflect calculated fits of data. Fitting were performed using the one set of binding site model.
0,5
7
0,0
6
0,0
5
(H / n(1)) / kJ mol
-1
-0,5
(H / n(1)) / kJ mol
-1
-1
(H / n(1)) / kJ mol
-0,5
-1,0
4
3
2
-1,0
-1,5
-1,5
1
0
-2,0
0,0
0,1
0,2
0,3
0,4
-2,0
0,0
0,0
n(1) / n(poly dAdT - poly dAdT)
0,1
0,2
0,3
0,4
0,1
0,2
0,3
0,4
n(1) / n(poly rA - poly rU)
n(1) / n(poly dA - poly dT)
8
4
6
(H / n(1)) / kJ mol
(H / n(1)) / kJ mol
-1
-1
3
4
2
1
0
0
0,0
2
0,1
0,2
0,3
0,4
0,0
n(1) / n(poly dGdC - poly dGdC)
0,1
0,2
0,3
0,4
n(1) / n(poly dG - poly dC)
Figure S1. ITC titrations of polynucleotides with the compound 1, (sodium cacodylate/HCl
buffer, pH = 5.0, c = 0.05 mol dm-3). Data points are the experimental injection heats while the
solid lines represent fitting to one set of binding sites model. The best fits gave values of ratio
n[bound compound] / [polynucleotide] = 0.1  0.05 for the most complexes, for easier comparison values
of log Ks are recalculated for fixed n = 0.1.
6
0
5
(H / n(2)) / kJ mol
(H / n(2)) / kJ mol
-1
-1
-1
-2
-3
4
3
2
1
-4
0
-5
0,0
0,1
0,2
0,3
0,4
0,0
n(2) / n(poly dAdT - poly dAdT)
0,1
0,2
0,3
0,4
n(2) / n(poly dA - poly dT)
Figure S2. ITC titrations of polynucleotides with the compound 2, (sodium cacodylate/HCl
buffer, pH = 5.0, c = 0.05 mol dm-3). Data points are the experimental injection heats while the
solid lines represent fitting to one set of binding sites model. The best fits gave values of ratio
n[bound compound] / [polynucleotide] = 0.1  0.05 for the most complexes, for easier comparison values
of log Ks are recalculated for fixed n = 0.1.
The equilibrium constant, binding stoichiometry and reaction enthalpy change were
determined directly from the experiment, while entropy contribution and Gibbs energy
change were calculated from the calorimetric data. It should be noted that relatively high
binding constants with low heat changes didn’t allow accurate analysis of thermodynamic
binding parameters, thus data given in Table S4 should be considered as estimations.
Table S4. Data parameters observed during fitting with the model one set of sites for ITC
titration of polynucleotide with the compound 1 (fixed N = 0.1)
log Ka
ΔrH/kJ mol-1
ΔrS/J K-1 mol-1
T ΔrS/kJ mol-1
ΔrG/kJ mol-1
p(dAdT)2
5.8
-2.1
103
31
-33
pdApdT
5.6
8.1
134
40
-32
p(dGdC)2
5.3
12.0
141
42
-30
pdGpdC
6.3
3.8
134
40
-36
pApU
5.4
-2.8
93
28
-31
CD experiments
4
4
3
2
2 +poly dG-dC - poly dG-dC
3
1 + poly dGdC - poly dGdC
2
1
1
CD [mdeg]
CD [mdeg]
0
-1
-2
poli (dG-dC)2
r = 0.05
r = 0.11
r = 0.21
r = 0.31
r = 0.42
r = 0.52
-3
-4
-5
-6
-7
-8
260
280
300
poly dG-dC - poly dG-dC
r = 0.05
r = 0.10
r = 0.20
r = 0.30
r = 0.41
r = 0.51
-2
-3
-4
-5
-6
-9
240
0
-1
320
-7
220
340
240
260
280
300
320
340
 / nm
 / nm
Figure S3. CD titration of polynucleotides poly dG-dC - poly dG-dC (c = 2.3  10-5 mol dm-3)
with 1 (LEFT) and 2 (RIGHT) at molar ratios r = [compound] / [polynucleotide] (pH = 5.0,
buffer sodium cacodylate, I = 0.05 mol dm-3).
2
2
2 + poly dA- poly dT
CD [mdeg]
CD [mdeg]
1 + poly dA- poly dT
0
poly dA-poly dT
r = 0.05
r = 0.1
r = 0.2
r = 0.27
-2
0
poly dA-poly dT
r = 0.11
r = 0.18
r = 0.24
-2
-4
-4
240
260
280
300
240
320
260
280
300
320
 / nm
 / nm
Figure S4. CD titration of polynucleotides poly dA - poly dT (c = 2.0  10-5 mol dm-3) with 1
(LEFT) and 2 (RIGHT) at various molar ratios r = [compound] / [polynucleotide] (pH = 5.0,
buffer sodium cacodylate, I = 0.05 mol dm-3).
CD [mdeg]
4
2
poly A-poly U
r = 0.09
r = 0.15
r = 0.20
r = 0.31
r = 0.43
r = 0.56
6
CD [mdeg]
poly A-poly U
r = 0.05
r = 0.16
r = 0.36
r = 0.45
r = 0.56
r = 0.75
6
4
2
0
0
1 + poly A- poly U
-2
-2
240
260
280
 / nm
300
320
2 + poly A- poly U
240
260
280
 / nm
300
320
Figure S5. CD titration of polynucleotides poly A - poly U (c = 1.4  10-5 mol dm-3) with 1
(LEFT) and 2 (RIGHT) at various molar ratios r = [compound] / [polynucleotide] (pH = 5.0,
buffer sodium cacodylate, I = 0.05 mol dm-3).
poly A
r = 0.08
r = 0.13
r = 0.20
r = 0.33
r = 0.43
r = 0.56
4
2
poly A
r = 0.1
r = 0.15
r = 0.2
r = 0.3
r = 0.4
r = 0.5
8
6
CD [mdeg]
CD [mdeg]
6
4
2
0
0
+
1+ poly AH - poly AH
-2
240
260
280
300
+
320
+
2 + poly AH - poly AH
-2
-4
340
240
260
280
300
320
 / nm
 / nm
Figure S6. CD titration of poly AH+ - poly AH+ (c = 9  10-6 mol dm-3) with 1 (LEFT) and 2
(RIGHT) at various molar ratios r = [compound] / [polynucleotide] (pH = 5.0, buffer sodium
cacodylate, I = 0.05 mol dm-3).
poly U
r = 0.07
r = 0.11
r = 0.22
r = 0.34
r = 0.40
r = 0.51
6
poly U
r = 0.05
r = 0.1
r = 0.15
r = 0.17
r = 0.23
r = 0.27
4
2
4
CD [mdeg]
CD [mdeg]
6
2
0
0
-2
2 + poly U
1 + poly U
-2
-4
240
240
260
280
300
320
340
+
260
280
300
320
 / nm
 / nm
Figure S7. CD titration of poly U (c = 3.9  10-5 mol dm-3) with 1 (LEFT) and 2 (RIGHT) at
molar ratios r = [1] / [polynucleotide] (pH = 5.0, buffer Na cacodylate, I = 0.05 mol dm-3).
1 Tumir, L.-M.; Grabar, M.; Tomić, S.; Piantanida, I., Tetrahedron. 2010, 66, 2501-2513.
2 W. Saenger, Principles of Nucleic Acid Structure, Springer-Verlag: New York, 1983; p. 226.
3 C. R. Cantor, P. R. Schimmel, Biophysical Chemistry, vol. 3,. WH Freeman and Co., San Francisco,
1980, 1109-1181.
4 Grabar Branilović, M.; Tomić, S.; Tumir, L.-M.; Piantanida, I. Mol. BioSyst. 2013, 9, 2051-2062.
5 a) J. D. McGhee and P. H. von Hippel, J. Mol. Biol., 1974, 86, 469; b) G. Scatchard, Ann. N. Y.
Acad. Sci., 1949, 51, 660