Modulation of DNA-polyamide interaction by β-alanine substitutions: A study of positional effects on binding affinity, kinetics and thermodynamics

Article abstract of DOI:10.1039/c4ob01456a

Hairpin polyamides (PAs) are an important class of sequence-specific DNA minor groove binders, and frequently employ a flexible motif, β-alanine (β), to reduce the molecular rigidity to maintain the DNA recognition register. To better understand the diverse effects that β can have on DNA-PA binding affinity, selectivity, and especially kinetics, which have rarely been reported, we have initiated a detailed study for an eight-heterocyclic hairpin PA and its β derivatives with their cognate and mutant sequences. With these derivatives, all internal pyrroles of the parent PA are systematically substituted with single or double βs. A set of complementary experiments have been conducted to evaluate the molecular interactions in detail: UV-melting, biosensor-surface plasmon resonance, circular dichroism and isothermal titration calorimetry. The β substitutions generally weaken the binding affinities of these PAs with cognate DNA, and have large and diverse influences on PA binding kinetics in a position- and number-dependent manner. The DNA base mutations have also shown positional effects on the binding of a single PA. Besides the β substitutions, the monocationic Dp group [3-(dimethylamino)propylamine] in parent PA has been modified into a dicationic Ta group (3,3′-diamino-N-methyldipropylamine) to minimize the frequently observed PA aggregation with ITC experiments. The results clearly show that the Ta modification not only maintains the DNA binding mode and affinity of PA, but also significantly reduces PA aggregation and allows the complete thermodynamic signature of eight-ring hairpin PA to be determined for the first time. This combined set of results significantly extends our understanding of the energetic basis of specific DNA recognition by PAs.

Full text of DOI:10.1039/c4ob01456a

Organic &
Biomolecular Chemistry
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PAPER
Modulation of DNA–polyamide interaction by
β-alanine substitutions: a study of positional
effects on binding affinity, kinetics and
thermodynamics†
Cite this: Org. Biomol. Chem., 2014,
2, 7523
1
a
b
b
b
b
Shuo Wang, Karl Aston, Kevin J. Koeller, G. Davis Harris Jr., Nigam P. Rath,
b
a
James K. Bashkin* and W. David Wilson*
Hairpin polyamides (PAs) are an important class of sequence-specific DNA minor groove binders, and fre-
quently employ a flexible motif, β-alanine (β), to reduce the molecular rigidity to maintain the DNA recog-
nition register. To better understand the diverse effects that β can have on DNA–PA binding affinity,
selectivity, and especially kinetics, which have rarely been reported, we have initiated a detailed study for
an eight-heterocyclic hairpin PA and its β derivatives with their cognate and mutant sequences. With
these derivatives, all internal pyrroles of the parent PA are systematically substituted with single or double
βs. A set of complementary experiments have been conducted to evaluate the molecular interactions in
detail: UV-melting, biosensor-surface plasmon resonance, circular dichroism and isothermal titration
calorimetry. The β substitutions generally weaken the binding affinities of these PAs with cognate DNA,
and have large and diverse influences on PA binding kinetics in a position- and number-dependent
manner. The DNA base mutations have also shown positional effects on the binding of a single PA.
Besides the β substitutions, the monocationic Dp group [3-(dimethylamino)propylamine] in parent PA has
been modified into a dicationic Ta group (3,3’-diamino-N-methyldipropylamine) to minimize the fre-
quently observed PA aggregation with ITC experiments. The results clearly show that the Ta modification
not only maintains the DNA binding mode and affinity of PA, but also significantly reduces PA aggregation
and allows the complete thermodynamic signature of eight-ring hairpin PA to be determined for the first
time. This combined set of results significantly extends our understanding of the energetic basis of
specific DNA recognition by PAs.
Received 30th May 2014,
Accepted 31st July 2014
DOI: 10.1039/c4ob01456a
www.rsc.org/obc
5
,6
new applications of PAs. Much of the advancement made
with PAs is due to the research on DNA recognition rules for
Introduction
Polyamides (PA) are N-methylpyrrole (Py) and N-methylimid- stacked heterocycle-pairs for hairpin PAs developed by Dervan
3
,7,8
azole (Im) based cations that derive from the natural product and coworkers.
AT base-pair specific minor groove binders distamycin and ficity achieved through covalent linking of PAs into hairpin
The enhancement of PA affinity and speci-
1
–4
netropsin.
stacked dimer in wider DNA minor grooves and recognize both lent, stacked dimers.
strands of the double helix led to extensive developments and number of heterocycles, they go out of register with the DNA
The discovery that distamycin could form a structures was a major improvement over the original noncova-
7
,9
However, as PAs increase in the
1
0,11
base pairs, and both affinity and selectivity are lost.
To
overcome this size and rigidity barrier, flexible inserts such as
a β-alanine (β) in place of one or more heterocycles can help us
to reset the heterocycle-base pair register. A number of
studies have indicated that β inserts can improve the
affinity and specificity of hairpin PAs for their target DNA
a
Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State
University, Atlanta, GA 30303, USA. E-mail: wdw@gsu.edu; Fax: +1 404-413-5505;
Tel: +1 404-413-5503
b
Department of Chemistry & Biochemistry, Center for Nanoscience, University of
Missouri-St. Louis, St. Louis, MO 63121, USA. E-mail: bashkinj@umsl.edu;
1
2–15
Fax: +1 314-516-5342; Tel: +1 314-516-7352
†
sequence.
Electronic supplementary information (ESI) available: Experimental details and
There are, however, some questions about optimum posi-
tioning for substitution of Py by β inserts and depending on
the DNA and PA sequences, β substitution can decrease the
characterization data (Tables S1–S3 and Fig. S1–S9) and X-ray crystallographic
information (Tables X-ray 1–18 and Fig. X-ray 1–6). CCDC 993654, 993655 and
9
93894. For ESI and crystallographic data in CIF or other electronic format see
1
6–19
DOI: 10.1039/c4ob01456a
binding affinity of PA molecules for DNA target sites.
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Fig. 1 Parent polyamide, KA1033, and both β- and Ta-containing analogs along with their cognate DNA sequence are shown. Internal pyrroles are
shown in blue and β inserts in red.
The role of β in DNA complex formation by PAs is thus more position-dependent effect of single and double β substitution
complicated than anticipated. Clearly, it is essential to under- placement on PA–DNA binding affinity and kinetics.
stand the influence of the number and position of β substi-
There is, however, an additional dimension to the kinetics
tutions on PA affinity and specificity for rational design of the and thermodynamics of PA interactions, namely strong PA–PA
most effective, biologically active agents. The binding affinity interactions that are independent of DNA binding, including
and selectivity of hairpin PAs with their target sequences have intermolecular hydrogen bonding that involves amide NH-
been extensively investigated by several groups using footprint- groups and acceptors such as carbonyl oxygens, and π–π stack-
1
6,20–23
ing and other methods.
However, the kinetics and ing of the pyrrole and imidazole rings. These interactions,
detailed thermodynamics of PA–DNA interaction have received perhaps enhanced by the flat, crescent shape of PA segments,
1
3,18,24,25
28,29
much less attention.
To aid in establishing the diverse can lead to aggregation.
Aggregation of hairpin PAs is
effects of β inserts, we have initiated a detailed study of the dependent on a number of parameters not described in the
binding thermodynamics, selectivity and kinetics for an eight- literature, but which we have been characterizing, and com-
heterocyclic PA, KA1033, and five β-containing derivatives with pounds similar to or larger in size than KA1033 are particularly
a DNA recognition sequence of the human COX2 promoter, prone to aggregate (unpublished work by us and colleagues
5
′-TTGGAGA-3′ (Fig. 1). Among the five derivatives, three C. M. Dupureur and M. R. Nichols). As shown below, even in
(KA1011, KA1013 and KA2127) have a single β substitution in the crystal structures of dimeric PA building blocks, the hydro-
different positions, and the other two (KA2128 and KA2129) gen bonding responsible for much of the aggregation is
have double βs in the positions covering the single β inserts. readily seen as intermolecular interactions. In an effort to
We note that the PA KA1033 and an alkylator attached analog minimize PA aggregation and obtain thermodynamics of PA–
of KA1013 were evaluated with other DNA sequences in early DNA binding, we increased charge–charge repulsion between
2
6,27
studies of PA–DNA interaction.
With these derivatives, all PA molecules by replacing the monocationic Dp group (3-(di-
internal Py positions of the parent PA KA1033 are substituted methylamino)propylamine) in KA1033 with the dicationic Ta
with β groups (Fig. 1). Five mutant DNA sequences with one or group (3,3′-diamino-N-methyldipropylamine) in KJK6021. Two
two base pair mutations in different positions have also been PAs having the same structure as KA1033 and KJK6021 but
studied to evaluate the positional role or sequence context of different anionic counterions, KJK6053 and KJK6064, have also
DNA base pairs in the PA–DNA interactions. Both PA modifi- been synthesized and studied. The results presented here
cations and DNA base mutations show that there is a large show that the counterions used do not affect the PA binding
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affinity and thermodynamics with cognate and mutant DNAs which has a β substitution just next to that in KA2127, has a
under our conditions. Modification of Dp to Ta, however, not significantly lower ΔTm (2.0 °C). Two double β-substituted PAs,
only maintains the DNA binding affinity and binding mode of KA2128 (ImImβ
PAs, but also significantly reduces PA aggregation. Presumably, γ-PyPyβPy-β-Ta), have much lower ΔT
due to aggregation of eight-ring PAs, no ITC experiments have parent. The ΔTm value for KA2129 is approximately the average
̲
̲
̲I m-
̲
m
2
9
been reported for PAs of this size. The Ta substitution allows of its two single β analogs KA1011 and KA1013. But the ΔT
m
the eight-ring PA–DNA binding enthalpy to be determined for KA2128 is lower than either of its single β analogs KA1013
through ITC and allows the very first complete thermodynamic and KA2127.
signature of eight-ring hairpin PA–DNA binding to be deter- For the five mutant sequences embracing either single or
mined. The results significantly extend the understanding of double base pair mutations in different positions, PAs display
the molecular basis of specific DNA recognition by PAs.
quite diverse binding affinities as indicated by the significantly
changed ΔT values (Table 1). In order to obtain a more clear
comparison of binding, the ΔT values have been classified
with four colors (red, orange, green and blue), and the color
for each ΔT is given based on the value relative to the ΔT
with cognate DNA: <50% (red), 50%–80% (orange), 80%–100%
green), and >100% (blue) of the ΔTm for PA-cognate DNA
m
m
Results
m
m
Qualitative comparison of PA binding affinity by thermal
melting study
(
Thermal melting provides a qualitative and relative compari- complex. It is clear that all PAs bind more weakly to mutants
3
0
son of compound binding affinities with DNA. KA1033 (Im- 1–3, while PA complexes with mutants 4 and 5 have both
ImPyIm-γ-PyPyPyPy-β-Dp, Fig. 1) is an eight-ring hairpin PA, weakening and enhancing effects. The unexpectedly large
while KJK6021 has the same heterocycle sequence but with a sequence-dependent variations of the ΔT
Ta group replacing the Dp of KA1033 at the C-terminal tail. cussed in detail below.
The binding affinities of these two PAs and five β-substituted
m
values are dis-
Quantitative determination of PA binding affinity and kinetics
analogs (Fig. 1) with their cognate (5′-TGGAGA-3′) and five
by biosensor-surface plasmon resonance (SPR)
mutant DNA sequences (Fig. S1A†) were compared by thermal
melting in a 1 : 1 molar ratio. The normalized DNA melting Biosensor SPR is a sensitive technique to monitor the progress
curves with and without PAs are shown in Fig. S1B.† The ΔT of reactions in real time. It provides kinetics and equilibrium
values, the Tm of the PA–DNA complex minus the Tm of free binding affinities as well as stoichiometries and cooperativity
m
3
1,32
DNA for each PA with each DNA are listed in Table 1.
values of biomolecular interactions.
To quantitatively
For the cognate sequence (TGGAGA), the parent PA, evaluate the interactions of hairpin PAs with DNA and also to
KA1033, increased the DNA Tm by 7.8 °C, and the ΔTm for provide details of the positional effects of the internal β substi-
KJK6021, the Ta group containing analog, is slightly higher. tutions, SPR experiments were performed for KA1033 and
Three single β-substituted PAs increased the DNA T
different amounts: KA1013 (ImImβIm-γ-PyPyPyPy-β-Dp), which constants obtained from global kinetic fits (Fig. 2A) and the
has a β before the γ loop, has a lower ΔT than KA1033 equilibrium binding constants (K ) measured from both
5.4 °C), while KA2127 (ImImPyIm-γ-PyβPyPy-β-Ta), which has global kinetic and steady state fits (Fig. 2B) of the sensorgrams
a Ta tail and a β in the same stacking position as KA1013, but for different PAs are listed in Table 2. The K values measured
m
by very derivatives with their target DNA, TGGAGA. The kinetic rate
̲
m
D
(
̲
D
after the γ loop (Table 1), has a slightly lower ΔT
m
(8.0 °C) by both fits are quite comparable and the values measured by
than the parent with Ta. KA1011 (ImImPyIm-γ-PyPyβ
̲
a
m
Table 1 ΔT values of PAs with cognate site TGGAGA and five mutant sequences
a
The error of these ΔT
m m
values is ±0.2 °C based on experimental reproducibility. The color for ΔT is given based on the comparison with the
value for the PA-cognate DNA complex: <50% (red), 50%–80% (orange), 80%–100% (green), and >100% (blue) of ΔT
complex.
m
for the PA-cognate DNA
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Fig. 2 (A) SPR sensorgrams (color) and global kinetic fits (black overlays) for KA1033 and derivatives with their cognate DNA TGGAGA at 25 °C with
0 mM NaCl. (B) Steady state fits for SPR sensorgrams.
5
KA1033, the parent PA, has a very strong binding affinity
with the cognate sequence (KD = 2.5 ± 0.4 nM). This strong
Table 2 SPR analysis of kinetic rate constants and equilibrium affinities binding can be attributed to the fast association (k ) and slow
a
for PAs binding to their cognate site TGGAGA at 25 °C with 50 mM dissociation rates (k ) (Table 2). In the following discussion, all
d
a
m
NaCl . ΔT values with cognate DNA are included for comparison
comparisons are made with the properties of KA1033 unless
explicitly stated otherwise. An analogue eight-ring PA with a Ta
tail, KJK6021, has around four times lower k and k , and a
similar KD (2.0 ± 0.2 nM) to KA1033. These results indicate
that the replacement of the Dp group by a Ta influences the
kinetics, but does not significantly affect the overall binding
affinity due to k and kd compensation. The single β substi-
tution of the Py on the N-terminal side of KA1033 results in a
slightly lower k and a higher k , with a 5-fold decrease in the
binding affinity of KA1013 with the cognate DNA (KD = 12.1 ±
Kinetics
D
K (nM)
a
d
6
k
M
a
(×10
s
k
d
Kinetic
fit
Steady
state
ΔT
m
−
1
−1
(×10 s−1
−3
PA
)
)
(°C)
KA1033
KA1011
KA1013
KA2127
KA2128
KA2129
KJK6021
4.7 ± 0.7
2.6 ± 0.3
3.7 ± 0.4
1.8 ± 0.3
0.7 ± 0.2
1.1 ± 0.2
1.3 ± 0.2
12 ± 1
96 ± 6
45 ± 7
4.7 ± 0.3
13 ± 2
29 ± 5
2.5 ± 0.4
36.4 ± 0.4
12.1 ± 0.4
2.6 ± 0.2
18.6 ± 0.8
26.3 ± 2.2
2.0 ± 0.2
2.8 ± 0.3
36.6 ± 0.1
10.3 ± 0.5
2.9 ± 0.2
17.5 ± 0.1
24.0 ± 0.5
2.2 ± 0.3
7.8
2.0
5.4
8.0
3.6
3.2
8.8
a
a
d
2.6 ± 0.2
0
.4 nM). For KA1011, the single β substitution on the C-term-
inal side of the γ loop not only slows its association, but also
a
Errors listed are the standard errors for the fits.
7526 | Org. Biomol. Chem., 2014, 12, 7523–7536
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Fig. 3 CD spectra for TGGAGA with all PAs at 25 °C. Molar ratios of PA to DNA hairpin vary from 0 to 2.0 as the induced CD signal for the PAs
increases.
3
4
increases its dissociation by 8-fold and therefore causes a binding mode as expected for hairpin PAs. The titrations
5-fold weaker binding affinity (KD = 96 ± 6 nM). KA2127, were conducted from 0 : 1 to 2 : 1 molar ratios of PA to hairpin
another single C-terminal β-substituted PA, has both faster k DNA. The signal and pattern of the induced CD by KJK6021
1
a
and kd and a KD (2.6 ± 0.2 nM) comparable to KJK6021. are very similar to KA1033 which suggests that these two PAs
The results of these three PAs show that a single β inserted target the DNA minor groove in a very similar manner. This
at different positions can have diverse effects on PA binding again indicates that the change of Dp to Ta does not signifi-
kinetics, but in this PA sequence and for the cognate DNA cantly affect the PA binding affinity or the structural influence
target, β generally accelerates PA dissociation and weakens the of PA on DNA.
PA binding affinity.
The two double-β-substituted PAs, KA2128 and KA2129,
have Ta instead of Dp groups at the C-terminus. Relative
to their eight-ring analog KJK6021, both KA2128 and
KA2129 have slightly slower association with DNA but much
faster dissociation and around 10 times weaker DNA-binding
Thermodynamic study of PA–DNA binding
Effects of salt concentration and temperature. From the
qualitative thermal melting studies and quantitative SPR
experiments to CD spectra evaluation, the Ta group containing
affinities (Table 2). KA2129 has an average affinity (K
D
= 26.3 ±
KJK6021 exhibits binding similar to KA1033. However, the
replacement of the monocationic Dp by a dicationic Ta group
increases the number of positive charges of PA. According to
the counterion condensation theory, the charges of minor
groove binders affect the release of counterions on binding to
2
.2 nM) of its corresponding single β analogs, KA1011 and
KA1013, while the affinity of KA2128 is weaker than either of
the two single β analogs (KA1013 and KA2127). The order of
binding affinity obtained by SPR is in good agreement with
that from the DNA melting study. All of the experiments have
been repeated twice, and the results are reproducible within
experimental errors.
2
9,35
DNA.
In order to evaluate the thermodynamic signatures
for PAs with different charges, SPR experiments were con-
ducted for KA1033 (+1) and KJK6021 (+2) with their cognate
sequence TGGAGA under several salt concentrations at 25 °C.
The corresponding sensorgrams are shown in Fig. 4A, and the
rate constants and equilibrium constants determined from
Evaluation of the PA binding mode and DNA structural
changes by CD
CD spectroscopy monitors the asymmetric environment of 1 : 1 global kinetic fits of the sensorgrams are listed in Table 3.
ligands when they bind to DNA and thus can provide infor- Overall, as the salt concentrations increase, the k decreases
decreases for both PAs,
a
3
3
d D
mation about the ligand binding mode. In Fig. 3, the CD while the k increases and thus the K
3
6
results for the PA–DNA complexes are characterized by large, as expected. The KD values for KJK6021 under different salt
positive induced signals from around 300 to 370 nm, where concentrations are quite comparable with those for KA1033,
the PAs absorb and DNA signals do not interfere. The large but the k and k of KJK6021 binding are both much lower
a d
positive induced CD signals indicate a DNA minor groove than those for KA1033.
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Fig. 4 (A) SPR sensorgrams (color) and global kinetic fits (black overlays) for KA1033 and KJK6021 with cognate DNA TGGAGA at 25 °C as a function
of salt concentration. (B) Salt concentration dependence of kinetic constants and binding affinities for KA1033 and KJK6021. All values are calculated
based on data in Table 3.
Table 3 SPR analysis of kinetic rate constants and equilibrium affinities for KA1033 and KJK6021 binding to TGGAGA at 25 °C with different salt
a
concentrations
KA1033
KJK6021
(×10 M s−1
6
−1
)
k
d
(×10−3 s−1
)
K
D
(nM)
k
a
(×10 M s−1
5
−1
)
k
d
(×10−3 s−1
)
[
NaCl] (mM)
k
a
D
K (nM)
5
1
2
4
0
4.7 ± 0.7
2.9 ± 0.4
1.8 ± 0.3
1.1 ± 0.3
12 ± 1
13 ± 2
15 ± 2
17 ± 1
2.5 ± 0.4
4.5 ± 0.4
8.7 ± 0.5
16 ± 0.8
13 ± 2
2.6 ± 0.2
3.1 ± 0.2
3.9 ± 0.6
4.9 ± 0.5
2.0 ± 0.2
3.9 ± 0.3
9.0 ± 0.5
16 ± 0.4
00
00
00
8.0 ± 0.5
4.3 ± 0.3
3.0 ± 0.3
a
Errors listed are the standard errors for the 1 : 1 global kinetic fits.
The logarithm values for k , k and K for both PAs have by the counterion condensation theory for one charge inter-
a
d
D
3
6,37
been plotted as a function of salt concentration in Fig. 4B. The action,
and as previously observed on binding of another
2
9
linear changes of the log values for KA1033 [slopes = −0.72 for monocationic hairpin PA (KA1039). The slopes of the linear
log(k ), +0.18 for log(k ) and +0.91 for log(K )] are as predicted change for KJK6021 are −0.72, +0.31 and 1.04 for log(ka),
a
d
D
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Fig. 5 SPR sensorgrams (color) and global kinetic fits (black overlays) for KA1033 and KJK6021 with cognate DNA TGGAGA at different tempera-
tures. The SPR-determined binding affinities and kinetics are listed in Table 4.
log(k
dencies of k
kd is slightly more salt concentration dependent than KA1033
d
) and log(K
D
), respectively. The salt concentration depen- temperature independent, and quite large and negative
for KJK6021 are similar to KA1033, but its (−11.0 ± 0.2 kcal mol− ).
Enthalpy of binding. Besides the effects of salt concen-
slope 0.31 vs. 0.18). Based on counterion condensation tration and temperature on the kinetics and affinity of PA
1
a
and K
D
(
D
theory, the slopes of log(K ) indicate that approximately one binding, the determination of binding enthalpy is very critical
cation has been displaced by both PAs on DNA binding. The for a more detailed energetic understanding of the PA–DNA
number of phosphate contacts (Z) between PAs and DNA interactions. However, as previously observed, the eight-ring
can be determined by the slope/Ψ, where Ψ is the fraction of hairpin PAs aggregate under typical ITC standard concen-
phosphate shielded by condensed counterions and is 0.88 for trations, such as 50 µM, and generally prohibit the binding
3
5
17,29
double stranded B-DNA: Z = 0.91/0.88 = 1.03 for KA1033 enthalpy assessment.
and Z = 1.04/0.88 = 1.18 for KJK6021. Thus there is about one concentrations, down to 1.5 µM, have been used in the ITC
phosphate affected by binding both PAs. experiments for KA1033, KJK6021 and KA2127. The ITC titra-
Therefore, several quite low ligand
The effects of temperature on PA binding affinity and kine- tions for the three PAs are shown in Fig. S2–4,† and the
tics have also been investigated by SPR experiments. The sensor- binding enthalpies (ΔH) obtained at each PA concentration are
grams of KA1033 and KJK6021 with 200 mM NaCl–HEPES listed in Table S1 ESI,† and are plotted versus the reciprocal of
buffer from 18 °C to 40 °C are shown in Fig. 5. This salt con- PA concentrations in Fig. 6. For these three PAs, the ΔH values
centration was chosen due to both PAs having stronger first become much more negative, and then reach a constant
binding affinity than under higher salt concentration, and value as the PA concentrations decrease. The ΔH values for
fewer mass transfer limitations than under lower salt concen- KA2127 and KJK6021 at each concentration are generally more
trations with SPR experiments. The kinetic rate constants and negative than those for KA1033, and reach a plateau (−13.0 ±
−
1
−1
equilibrium constants determined from 1 : 1 global kinetic fits 0.4 kcal mol and −14.1 ± 0.2 kcal mol , respectively) at 0.4
−
1
of the sensorgrams are listed in Table 4. Based on the equili- µM (2.5 µM). The ΔHs obtained from 2.5 μM, 2.0 μM and
brium binding constants, the binding Gibbs free energy (ΔG) 1.5 μM (0.4 µM , 0.5 µM and 0.67 µM ) of KA2127 and
−
1
−1
−1
at different temperatures was also calculated (ΔG = −RT ln K , KJK6021 agree very well within experiment error and indicate
eq
−
1
−1
where R is 1.987 cal mol
K
and T is 298 K). The data clearly that we have obtained the actual binding enthalpies. The ΔHs
−1
show that as the temperatures increase, both k
a
and k
d
for KA1033 are consistent (−11.8 ± 0.3 kcal mol ) at 2.0 µM
increase, and the overall binding affinity K decreases for both and 1.5 µM (0.5 µM− and 0.67 µM ), which suggests that the
1
−1
D
PAs. However, ΔG values for both PA bindings are essentially actual binding enthalpy for KA1033 has also been reached
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Table 4 SPR analysis of kinetic rate constants and equilibrium affinities as a function of temperature for KA1033 and KJK6021 binding to TGGAGA
a
with 200 mM NaCl
KA1033
(×10 M s−1
6
−1
)
k
d
(×10−3 s−1
)
K
D
(nM)
ΔG (kcal mol−1
)
Temp. (°C)
k
a
18
25
32
40
1.4 ± 0.2
1.8 ± 0.4
2.2 ± 0.3
3.0 ± 0.6
6.9 ± 0.3
15 ± 2
30 ± 4
5.0 ± 0.4
8.7 ± 0.5
14 ± 0.5
18 ± 0.8
−11.1
−11.0
−11.0
−10.8
52 ± 6
KJK6021
(×10 M s−1
5
−1
)
k
d
(×10−3 s−1
)
ΔG (kcal mol−1
)
Temp. (°C)
k
a
D
K (nM)
18
25
32
40
2.9 ± 0.3
5.0 ± 0.4
8.7 ± 0.6
18 ± 1
2.0 ± 0.2
4.5 ± 1.0
11 ± 2
6.8 ± 0.5
9.0 ± 0.4
13 ± 0.8
14 ± 0.06
−10.9
−11.0
−11.0
−11.0
25 ± 4
a
Errors listed are the standard errors for the 1 : 1 global kinetic fits.
Table 5 Thermodynamic profiles for KA1033, KJK6021 and KA2127
a
with their cognate DNA TGGAGA with 50 mM NaCl at 25 °C
ΔG (kcal mol−
1
)
ΔH (kcal mol−1
)
TΔS (kcal mol−1
)
KA1033
KJK6021
KA2127
−11.7
−11.9
−11.7
−11.8
−14.1
−13.0
−0.1
−2.2
−1.3
a
The TΔS are calculated using ΔG = ΔH − TΔS.
very well with those for their counterion analogs KA1033 and
KJK6021, and the corresponding ΔT values are also quite com-
m
parable for PAs of the same structure (KJK6053 vs. KA1033,
KJK6064 vs. KJK6021). ITC experiments have also been conducted
with 2.5 µM of KJK6053 and KJK6064 into 10 µM of cognate
DNA hairpin, and compared with the titrations of KA1033 and
Fig. 6 Binding enthalpies of different concentrations of KA1033, KJK6021
and KA2127 with 10 μM TGGAGA hairpin DNA measured by ITC at 25 °C.
KJK6021 at the same PA concentration (Fig. S6, ESI†). The ΔH is
−1
−
8.3 ± 0.3 kcal mol for KJK6053 binding and −14.0 ± 0.3 kcal
−1
mol for KJK6064 binding. Both of these values are consistent
with the ΔH values measured for KA1033 and KJK6021. These
results suggest that under our conditions (50 mM NaCl in the
buffer) the formate and TFA counterions do not significantly
affect the affinity and thermodynamics of PA binding.
with these two concentrations. These results indicate that with
the decrease of sample concentration in ITC, concentration-
dependent PA aggregation can be reduced to an insignificant
amount. Once the PA aggregation has been totally overcome,
the actual binding enthalpy can be determined.
Based on SPR binding free energy values and the ITC
enthalpy values, the TΔS values were calculated from ΔG = ΔH
Besides the modification of Dp to Ta, the counterion used
during isolation of KA1033 (Dp) is also different from that for
−
TΔS for KA1033, KA2127 and KJK6021 (Table 5). Comparison
of the contributions of the enthalpy and entropy to the free
energy shows that PA complex formation is dominated by the
favorable binding enthalpies. This is the first complete ther-
modynamic study for DNA minor groove binding with eight-
ring hairpin PAs.
•
−
KJK6021 (Ta): trifluoroacetate ( CF COO , TFA) with KA1033
3
•
−
while formate ( HCOO ) is used for KJK6021. In order to evalu-
ate the effects of counterions on PA binding affinities and
thermodynamics, two PAs in the same structure as KA1033 or
KJK6021 but with switched counterions, KJK6053 (ImImPyIm-
γ-PyPyPyPy-β-Dp, formate) and KJK6064 (ImImPyIm-γ-PyPy-
PyPy-β-Ta, TFA), respectively, have been studied with UV-
melting experiments. The thermal melting curves for these
four PAs with cognate and mutant DNA sequences are shown
Discussion
Effects of DNA sequence and PA structure on relative binding
affinity
in Fig. S5,† and the ΔT
m
values are listed in Table S2, ESI.† The relative binding affinities of KA1033 and derivatives with
The melting curves for KJK6053 and KJK6064 complexes agree five mutant DNA sequences with either single or double base
7530 | Org. Biomol. Chem., 2014, 12, 7523–7536
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pair mutations in the PA binding site have been determined the higher ΔT
m
values than with cognate DNA. The flexibility
(
(
Table 1). Through the comparison of ΔTm values of KA1033 from the double β inserts might play a role in the preferred
ΔT = 7.8 °C) with mutants 1–3, it is clear that a mutation to binding for KA2128 and KA2129. The β-modulated binding
m
reverse the third base pair of the 5′-TGGAGA-3′ site (mutant 1, orientation of PA has also often been observed by Dervan and
3
8–40
5
′-TGC
stability much more significantly than the complete changing as previously found with other PA–DNA complexes, the
of the fourth base pair (mutant 2, 5′-TGGCGA-3′, ΔT different contributions of the base pairs in the targeting site
.9 °C), while the mutant at the fifth base pair (mutant 3, cause distinct affinity reductions for the β-substituted PAs and
′-TGGATA-3′, 6.9 °C) influences the binding the least. These different effects on binding affinities for the PA-bound DNA
̲
AGA-3′, ΔT = 0.6 °C) decreases the induced thermal coworkers using different powerful methods.
In summary,
m
1
7
̲
m
=
3
5
̲
mutation results are in agreement with the position-dependent mutants. Moreover, the possibility to modulate PA binding
effects of β substitution in PA, since KA1011, with β targeting orientation through incorporating double β inserts provides
the most sensitive third base pair, has a much lower ΔT than possible additional ideas for rational drug design. More
m
KA1013, with β targeting the less sensitive fourth base pair. studies of additional β substituted PAs will be required before
Moreover, the ΔTm values for all PAs with mutants 1–3 suggest we can define the complex rules for their effects on KD in
that all of these three single DNA base pair mutations can different DNA sequences.
rd
th
weaken the PA binding in the order: 3 base pair >4 base
pair >5 base pair.
th
Effects of Dp and Ta groups and internal β on PA–DNA
thermodynamics
In mutant 4 (5′-TGGT ̲G A-3′), the fourth base pair (A/T) of
the 5′-TGGAGA-3′ site has been switched into T/A, and KA1033, ITC titrations have been previously performed for another
KA1011, KA1013 and KA2129 display higher ΔTm values than eight-ring hairpin PA KA1002 and its single β-inserted analogs,
with cognate DNA. This result means that their Py/Py and β/Py but their binding enthalpies could not be determined, pre-
pairs which target the fourth base pair in the binding site sumably due to the significant compound aggregation at
1
7
favor T/A base pair recognition slightly more than the A/T base current ITC concentration requirements.
pair. In contrast, KA2127 has a slightly lower ΔT with mutant enthalpy of a six-ring hairpin PA was first published in 1996,
than with cognate DNA (6.7 °C vs. 8.0 °C) which suggests the and in the following eighteen years, there were no reports on
Py/β pair in KA2127 prefers to target the A/T base pair. The larger PAs. In the experiments reported here, we have been
ΔT values for KA2128 with cognate DNA and mutant 4 are able to determine direct ITC binding enthalpies for eight-ring
The binding
4
1
m
4
m
very similar (3.6 °C vs. 3.4 °C) which indicates that the β/β pair PA for the first time. KA1039, a six-ring hairpin derivative of
does not have a strong binding preference to the A/T or T/A KA1002, shows much less aggregation than KA1002 in ITC
1
4
base pair. These data clearly show that the incorporation of β and thus allows its ΔH to be determined by using diluted
1
7,29
inserts can have very diverse influences on the specificity of samples.
DNA recognition by PAs, and these effects strongly depend on obtain the thermodynamic signatures of eight-ring hairpin
the PA structure and DNA sequence. PAs, the monocationic Dp group in KA1033 has been modified
Here, in order to reduce the aggregation and
Mutant 5 has base pair changes at both the third and into a dicationic Ta (KJK6021), and studies on both full hetero-
fourth base pairs of the 5′-TGGAGA-3′ site (mutant 5, cycle and β-substituted (KA2127) hairpins have been con-
5
′-TGA̲G ̲G A-3′) by switching the middle GA to AG. These ducted (Fig. S2–4, ESI†). The results (Fig. 6) clearly confirm
mutations could either reverse the binding orientation of PAs that PA aggregation depends on compound charge and con-
or significantly weaken the PAs binding if they keep the orig- centration, and can be minimized as the PA concentrations
inal binding orientation, as discussed above with mutants 1 decrease and the charge increases. KA1033 shows a constant
and 2. Based on the recognition sequence, these results ΔH when its concentration is around 2 μM or less, while
suggest that the original orientation is the usual N → C (Im- KA2127 and KJK6021 reach a constant ΔH with concentrations
ImPyIm) for KA1033, for example, bound 5′ → 3′ to 5′-TGG- equal to or less than 2.5 μM. From the comparison of the
AGA-3′, while with mutant 5, binding becomes reversed, curves in Fig. 6 in the range where all PAs aggregate (10 μM–
with N → C (ImImPyIm) targeting 3′ → 5′ to 3′-AGGAGT-5′. It 2.5 μM), the ΔH of single β substituted PA KA2127 is always
should be noted that although the results are consistent with closer to the constant binding enthalpy than its full hetero-
this interpretation, more detailed experimental studies, such cyclic analog KJK6021. The observed ΔH of the Ta group con-
as NMR or X-ray analyses, would be necessary for a more taining KJK6021 is always closer to the actual value than the
definitive answer. The ΔT
m
values with mutant 5 for KA1033, Dp group containing KA1033. These data show that PAs aggre-
KJK6021 and their single β analogs, KA1011, KA1013 and gate in the order KA2127 < KJK6021 < KA1033, and suggest
KA2127, are all lower than the ΔTm for the cognate sequence, that additional positive charge from the Ta group and the extra
but greater than ΔT
the two base pair mutations altered the PA binding into the cular scale picture of the amide hydrogen bonds that we
reverse orientation (3′ 5′). Interestingly, two double believe, along with π-stacking, constitute strong tendencies
m
with mutants 1 and 2. This suggests that flexibility from β help to reduce PA aggregation. To see a mole-
→
β-substituted PAs, KA2128 and KA2129, display an opposite be- toward aggregation, the crystal structures of two dimer build-
havior in that they prefer mutant 5 over cognate binding and ing blocks are instructive (Fig. 7 and Fig. X-ray 2, X-ray 4 and
probably have the reverse binding orientation, as indicated by X-ray 6, ESI†). We clearly see from Fig. 7 that, for Boc-Py-
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cation KA1033 (Fig. 4B), and this is different from a classical
two charge interaction as predicted by the counterion conden-
3
5,36
sation theory.
The two positively charged amines in Ta are
connected on the same linker (Fig. 1) and, as a result of this
closer spacing, they may have strong electronic interactions
with only one phosphate on the DNA backbone, and thus,
release only one counterion on PA binding.
The SPR results for KA1033 and KJK6021 as a function of
temperature show that the temperature dependences of kine-
tics for KJK6021 binding are greater than that for KA1033
(
Fig. 5B). This suggests that the relatively longer Ta group
becomes more dynamic than the Dp tail as the temperature
increases, and contributes to the larger kinetic changes of
KJK6021. Interestingly, the changes in Ta kinetics are again
partially cancelled as a result of the temperature dependences
of binding affinities for KJK6021 and KA1033. The binding
free energies for both Dp and Ta complexes are also quite
similar and are essentially temperature independent in the
range that could be studied (Table 4). This signature of temp-
erature on DNA minor groove binding free energy has been
Fig. 7 Close-up view of intermolecular NH and OH hydrogen bonding
in the crystal structure of Boc-Py-β-COOH·0.5dioxane. The Boc-amine-
NH donates an H-bond to the carboxylic acid carbonyl of an adjacent
2
9
molecule, while the amide carbonyl accepts H-bonds from the observed with other minor groove complexes.
carboxylic acid OH group and amide NH of an adjacent molecule.
In summary, based on the UV melting data, CD titrations
and SPR studies, Dp and Ta groups have very similar effects on
DNA recognition by PAs, but the dicationic Ta group is capable
β-COOH·0.5dioxane, amide NH to carbonyl and related inter- of reducing PA aggregation in ITC experiments. In the pres-
molecular H-bonds dominate intermolecular interactions in ence of a Ta group, an additional β can further decrease the PA
the solid state. Structural details and more extensive aggregation (Fig. 6). The thermodynamic profiles (Table 5)
H-bonding views are provided in ESI.† This structure makes it suggest that complex formation of KA1033, KJK6021 and
easy to understand why the increasing size of PAs with associ- KA2127 on the TGGAGA site is strongly driven by enthalpy
ated increased intermolecular interactions can lead to PA which is opposite to most of the classic minor groove binders
4
4
aggregation. At about eight-ring PA size, the concentration for that target AT-rich sites. The presence of GC base pairs in
PAs to aggregation is lower than that used in ITC experiments, the target binding site changes the geometry and hydration
which has prevented a previous determination of eight-ring status of the minor groove relative to that of AT-rich sites, and
PA–DNA binding enthalpies.
the stacking of Im and Py rings and numerous H-bonds
To obtain a better understanding of the effects of counter- between PA–DNA convert minor groove binding thermo-
ions on PA binding affinity and thermodynamics, KJK6053 and dynamics from entropy driven with most of the AT-rich cases
KJK6064 have been investigated and compared with their into enthalpy driven. This is the first calorimetrically deter-
analogs KA1033 and KJK6021, which have the same structure mined binding enthalpy for eight-ring hairpin PAs and pro-
but different counterions. The quite comparable binding vides an increased understanding of PA–DNA interaction
affinities with cognate and mutant DNA sequences through thermodynamics, including a shift in enthalpic vs. entropic
UV-melting (Fig. S5, ESI†) and the constant binding enthalpies control of binding.
obtained through ITC (Fig. S6, ESI†) for these PAs suggest that
the counterions, TFA and formate, do not have a significant
influence on PA–DNA interaction under the experimental con-
Effects of β inserts and PA modification on binding affinity
and kinetics
ditions. However, it is still worthwhile to note that the counter- In order to extend our understanding of the effects of different
ions always play critical roles for the biological activities of numbers of internal β building blocks on DNA recognition,
4
2,43
synthetic small molecules.
comprehensive binding affinity studies of PAs were conducted
The effects of Dp and Ta groups on PA–DNA complex for- with SPR. The results show that β substitutions for hetero-
mation have also been systematically investigated as a function cycles in this sequence (Fig. 1) generally weaken the binding
of salt concentration by SPR. Under a wide range of salt con- affinities of PAs with their cognate DNA. Together with pre-
1
6,17
centrations (from 50 mM to 400 mM NaCl, Table 3), relative to vious studies of other eight-ring PA–DNA interactions
the
Dp, the more flexible Ta group slows down both association weakening effect that single and double β substitutions can
and dissociation rates of PA. But the effects on kinetics par- have on PA binding affinity is further confirmed. Moreover, the
tially cancel so that Ta gives only a small affinity increase. detailed evaluations in this work substantially demonstrate
Moreover, the salt concentration dependencies of kinetics and the large and diverse influences of β substitutions on the
affinity for the dication KJK6021 are quite similar to the mono- association and dissociation rates of PA–DNA interaction in a
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position-dependent manner (Table 2). The parent PA, KA1033,
is a strong binder with the highest association rate among all
Conclusions
PAs reported here, from which we infer an easily-optimized In conclusion, the energetic effects of positional substitution
shape for the minor groove binding site. PAs, especially those of Py by single and double βs on hairpin PA–DNA interaction
containing internal βs, have numerous additional confor- have been investigated in detail. As previously reported with
mations attainable in solution because of the possibilities for other PAs, reduced binding affinity has been observed for all
rotation about single bonds. The γ-turn and C-terminal groups of the β-modified PAs. The SPR results clearly illustrate that
also have several rotatable bonds, and Ta has more than Dp. the β substitution can have large, position- and number-depen-
However, on optimized binding DNA, PAs must adopt a less dent effects on association and dissociation rates, and also the
dynamic conformation with a crescent shape to match the binding affinities of PAs in the same DNA binding site. Com-
minor groove structure. In addition, stacked PA heterocycles parison of mutant DNA sequences with all PAs also shows the
and numerous H-bonds between PAs and DNA must be appro- position-dependent contributions of DNA base pairs in the PA
priately aligned in the minor groove complex. The necessary binding and the preference of binding orientation for PAs.
molecular rearrangements and alignments both require time A modification of the positively charged functional Dp group
and consequently affect the association rates of PAs. The k
a
into a Ta group, rather than a change in the heterocycles, pre-
values for single internal β-containing PAs, KA1011, KA1013 serves the PA binding mode and affinity but reduces PA aggre-
and KJK6021, are slower than that for all heterocycle-based gation in ITC. This made a thermodynamic study possible for
KA1033. In addition, the k values for double internal β-con- eight-ring hairpin PAs for the first time, and it took eighteen
a
taining PAs (KA2128 and KA2129) are even slower than that for years to advance our understanding of the energetic basis of
KJK6021. This can be explained by the increased flexibility of PA–DNA interactions from six-ring to eight-ring PA. All of the
the β group and the increased number of conformations in above information significantly extends our knowledge of PA–
solution that do not have a shape to match the DNA minor DNA complexes and takes our understanding of the kinetics
groove. Reorientation to bind DNA requires additional time and thermodynamics of DNA–PA molecular recognition an
relative to PAs with only heterocycles and thus slows the kine- important step forward.
tics of association. It should also be noted that another single
β-inserted PA, KA2127, has a slightly faster k than KJK6021,
a
which means that the presence of some β inserts can also
increase the PA association rates. In such cases, the added β
inserts must adjust in more rapid alignment with functional
Materials and methods
DNA and compounds
groups in the DNA minor groove. These very diverse effects DNA sequences were purchased from Integrated DNA Techno-
that β inserts can have on PA association rates might also cor- logies, Inc. (Coralville, IA, USA). The synthesis and verification
1
8
relate with the torsion angles of PA, as suggested by a study of of KA1011 and KA1013 has previously been reported, and
4
5
other eight-ring PAs from the Sugiyama group. Additional that of the other PAs in Fig. 1 are presented in ESI.† Synthesis
kinetics studies on β containing PAs will be required to estab- was generally based on the reported solid-phase Boc
4
6
lish the molecular basis for these differences.
approach. The main changes to standard procedures were
For the dissociation rates, the order for Dp containing PAs the use of PyBOP (benzotriazol-1-yl-oxy-tripyrrolidinophos-
is KA1011 > KA1013 > KA1033, and the order for Ta containing phonium hexafluorophosphate) as the coupling agent and the
PAs is KA2129 > KA2128 > KA2127 > KJK6021 (Table 2). Based extensive use of dimer building blocks. Dimer building blocks
on the decreasing order of dissociation rates, it is clear to see have long been used in solid and solution phase
1
0,46–48
that the double β inserts speed up the PA dissociation more chemistry,
but their use in solid phase PA chemistry has
than single β inserts. This is the first instance where the often been limited to cases where the poorly nucleophilic
number and position of β are shown to have such a large and amine of the imidazole amino acid does not couple well to an
4
6
diverse effect on association and dissociation rates and incoming active ester. We, however, used dimers at essen-
affinities of PAs in the same binding site. These results empha- tially every step in order to improve the solid phase yield and
size that the β group does not have the same favorable contacts cut the number of solid-phase coupling steps in half. Many of
with the minor groove as a Py heterocycle. For a β group to these dimers are commercially available, including Boc-Py-
increase the affinity of a PA for DNA, it must increase PyCOOH and Boc-β-PyCOOH, though they are not always avail-
other interactions in the PA, through resetting the PA–DNA able, so we provide several procedures for dimer syntheses and
interaction register, that are lost due to a switch of the hetero- scale-up, and characterization data where appropriate (see
cycle to a β. Additional studies of the correlation between ESI†). Furthermore, both Boc-Py-β-COOH and Boc-β-PyCOOH
4
9,50
β inserts and PA binding are in progress with other PA have appeared in the literature but were not characterized.
4
6
sequences and DNAs. Overall, all of the current results are One standard synthesis of dimers uses the water-soluble
sufficient to validate the various modulation capabilities that carbodiimide EDC, but we found that the much less expensive
the β group can have on PA binding affinity and kinetics, combination of carbonyldiimidazole (CDI) and imidazolium
5
1
which confirms the β group as an important module in PA hydrochloride described by Woodman et al. worked well to
design.
form the Boc-Py-β-COOH and Boc-β-PyCOOH dimers (both
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methods gave 60–70% isolated yields, much lower than the KJK6021. A flow rate of 75 µL min− is used in a KA1033 and
1
4
8
>
90% yields reported and reproduced in our lab for other KJK6021 comparison (Fig. 4 and 5). Kinetic analyses were per-
dimers with a range of coupling agents, but reagents for the formed by global fitting of the binding results for the entire
th
CDI method are 1/10 the cost of EDC). The use of the pre- concentration series using a standard 1 : 1 kinetic model with
formed HOBt ester Boc-Py-OBt has also been reported for integrated mass transport-limited binding parameters as
4
8
53,54
dimer synthesis, and this is a very efficient method; it gave a described previously.
9
Steady-state analyses were also con-
)/
7% yield of Boc-Py-β-COOH on a 20 g scale (see ESI† for scale- ducted and data were fit with a one site model: r = (Keq × C
f
up of Boc-Py-PyCOOH and Boc-Py). CDI and Boc-β-Ala-OH were (1 + Keq × C ), where r represents the moles of bound PA per
f
purchased from Chem-Impex Int’l (Wood Dale, IL, USA). Imid- mole of DNA hairpin duplex, Keq is equilibrium binding con-
azolium hydrochloride, β-alanine ethyl ester hydrochloride, stants, and C
f
is the free PA concentration in equilibrium with
dimethyl sulfoxide, trifluoroacetic acid, acetic anhydride, N,N- the complex. The observed steady-state response unit, RUobs
,
diisopropylethylamine, piperidine, 3-(dimethylamino)-1-propyl- at saturation of binding site divided by the calculated response
amine, and 3,3′-diamino-N-methyl-dipropylamine were pur- per bound PA, RUcal, gives the binding stoichiometry (n) of PA,
chased from Aldrich (St. Louis, MO, USA). Boc-Py-ImCO H, n = RUobs/RUcal
.
2
O
2
NPy-COCCl
3
and Boc-Py-OBt ester were from Oakwood
UV-thermal melting
Chemical (West Columbia, SC, USA). N,N-Dimethylformamide
and dichloromethane were from Applied Biosystems (Foster DNA melting (T ) curves were recorded with a Cary 300 UV
m
2
City, CA, USA). Boc-Py-PyCO H was from A. Chemtek (Worce- visible spectrophotometer (Varian Inc., Palo Alto, CA)
ster, MA, USA), Boc-γ-Abu-OH and PyBOP from Novabiochem equipped with a thermoelectrically controlled cell holder. The
1
(
Billerica, MA, USA), and Boc-β-PAM resin (0.23 meq g− ) from absorbances of the free DNAs (Fig. S1, ESI†) and PA–DNA com-
Peptides Int’l (Louisville, KY, USA). Boc-β-ImCO H was pre- plexes were measured at 260 nm from 25 °C to 95 °C with a
pared via PyBOP coupling/saponification according to the lit- heating rate of 0.5 °C min− . The DNA concentration was 3 µM
2
1
4
8
10
erature as was desIm-ImCO H.
in hairpin, and PA in a 1 : 1 molar ratio of DNA was added. The
m
T curves were normalized to give equimolar DNA concen-
2
X-ray crystal structures of the Boc-Py-β-COOH and Boc-
β-PyCOOH dimers (as dioxane and methanol solvates and as trations. All the thermal melting experiments were conducted
the dioxane solvate) were obtained as a possible aid to mole- three times with HEPES buffer containing 10 mM HEPES,
cular mechanics parameters, and ended up being quite 50 mM NaCl and 1 mM EDTA at pH 7.4.
instructive regarding intermolecular interactions (see the text
Circular dichroism (CD)
below on aggregation, and ESI†). New compounds were charac-
terized by elemental analysis, HPLC/MS, HRMS, and NMR, CD spectra were collected using a Jasco J-810 spectrometer
1
13
including H, C and 2-D methods. We changed from trifluoro- (Jasco Inc., Easton, MD) from 400 nm to 230 nm at 25 °C. The
acetate (TFA) salts to formate salts by changing the running spectra were averaged over four scans with a scan speed of
4
6
−1
buffer of the standard chromatographic methods
in preparative HPLC from 0.1% trifluoroacetic acid to 0.2% tion was first scanned and the PAs, at increasing concentration
used 50 nm min and a buffer blank correction. A 5 µM DNA solu-
formic acid.
ratios, were then titrated into the same cuvette and the com-
plexes were scanned under the same conditions. The same
hairpin DNA and buffer as those in the melting study were
Biosensor-surface plasmon resonance (SPR)
SPR measurements were performed with four-channel Biacore used.
000 and BiaT200 optical biosensor systems (GE Healthcare,
Inc., Piscataway, NJ). The process of DNA immobilization
2
Isothermal titration calorimetry (ITC)
3
1,52
has previously been described.
Briefly, a 5′-biotin labeled ITC experiments were performed using a MicroCal VP-ITC (GE
hairpin duplex containing PA binding site (5′-biotin- Healthcare, Inc., Piscataway, NJ) with VP-2000 software for
CCTTGGAGAGT CTCTCCAAGG, Fig. 1) was immobilized instrument control and Origin 7.0 for data analysis. The
̲T ̲T ̲T ̲
onto an SA chip. Filtered and degassed HEPES buffer contain- sample cell was filled with 10 µM of target DNA TGGAGA in
ing 10 mM HEPES, 1 mM EDTA and varying NaCl (from HEPES buffer, and 20 injections of 10 µL of PA solution at vari-
5
0 mM to 400 mM) at pH 7.4 with 0.05% v/v surfactant P20 able concentrations were performed incrementally. A delay of
was used in SPR experiments. 300 s was used between injections to ensure the equilibration
For the determination of PA binding affinity and kinetics, a of the baseline. The heat for each injection was obtained by
series of PA samples in different concentrations (1 nM to integration of the peak area as a function of time. The heats of
1
µM) was injected over the DNA sensor-chip until a steady dilution, determined by injecting compounds into the sample
state was obtained. Then, PA-free experimental buffer was cell containing only the buffer, were subtracted from those in
flowed to measure PA dissociation and this was followed by PA/DNA titrations to obtain the corrected binding-induced
chip regeneration with a pH 2.5 glycine solution, and baseline enthalpy changes. Because all the PAs bind quite strongly to
stabilization for the next cycle with multiple buffer injections. the cognate sequence in this work, the heat/mole of added
The flow rate of the entire cycle for all PAs in Fig. 2A is 25 µL compound is essentially constant in the initial titration region
−
1
−1
−1
min , but 100 µL min for KA2127 and 75 µL min for where all of the added compound is bound to DNA. The ΔH
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can be determined by a linear fit as shown in Fig. S2–4.† 19 K. J. Koeller, G. D. Harris, K. Aston, G. He,
HEPES buffer containing 10 mM HEPES, 50 mM NaCl and
mM EDTA at pH 7.4 was used for ITC experiments.
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