Threading intercalation
Undergraduate Category: Physics Degree Level: Bachelor Abstract ID# 633
The role of the threading moiety in DNA threading intercalation by ruthenium dimer complexes Andrew
1 Clark ,
Thayaparan
1 2 Paramanathan ,
3 Westerlund ,
Fredrik
Per
3 Lincoln ,
1 McCauley ,
Micah J.
Ioulia
4 Rouzina ,
and Mark
1 C.Williams
1Department
of Physics, Northeastern University, Boston, USA. 2 Department of Physics, Bridgewater State University, Bridgewater, USA. 3 Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden. 4Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, USA.
Fitting to McGhee von Hippel Isotherm
Threading intercalation
There is significant interest in ruthenium dimer complexes due to their novel DNA binding properties. The binuclear ruthenium complex ΔΔ-[μ-bidppz(bipy)4Ru2]4+ that we investigate in this study binds to DNA by threading intercalation, in which one of the bulky ruthenium moieties threads through the DNA base pair stack to reach its bound state. The extremely slow dissociation from DNA of this threading intercalator gives it properties needed for chemotherapy drugs, while also making it difficult to study in traditional bulk experiments. In this study, we use optical tweezers in order to quantify the threading kinetics, as well as the equilibrium behavior of this ruthenium dimer complex to understand how changing the ancillary ligands coordinated to ruthenium (threading moiety) affects the process of threading. To observe the slow threading process, the DNA was held at a constant force in the presence of the ligand and the DNA extension was measured as a function of time until the extension reached equilibrium. When the concentration of the ligand is increased during these constant force measurements, the total extension of the DNA will also increase until it becomes saturated at some concentration as all of the ligand binding sites are filled. These measurements of the equilibrium DNA extension allow for a full quantitative description of the binding kinetics and equilibrium behavior of the complex. The preliminary data suggests that the ΔΔ-[μ-bidppz(bipy)4Ru2]4+ complex with its bicyclic bipyridine ligand, shows significantly faster kinetics compared to the ΔΔ-[μbidppz(phen)4Ru2]4+ , complex with a tricyclic phenanthroline ligand counterpart.
The fractional equilibrium binding of ΔΔ-B (right, blue triangles) is estimated by,
During threading intercalation, the molecule must thread a large bulky moiety through dsDNA so that the central planar section can be stacked between the base pairs.
𝑥𝑒𝑞 𝐹, 𝐶 − 𝑥𝑊𝐿𝐶 (𝐹) 𝛩 𝐹, 𝐶 = 𝑥𝑠𝑎𝑡 𝐹 − 𝑥𝑊𝐿𝐶 (𝐹)
In order to dissociate from this state, the ligand has to unthread one of its bulky moieties from the DNA.
This was then fit to the McGhee von Hippel isotherm (right, red).
This results in high DNA binding affinity with extremely slow kinetics.
𝐶 𝑛 ∙ (1 − 𝛩)𝑛 𝛩 𝐾𝑑, 𝑛 = ∙ 𝐾𝑑 (1 − 𝛩 + 𝛩)𝑛−1 𝑛
Paramanathan, T. et. al. JACS 2008, 130, 3752-3753
Semi-rigid ruthenium threading intercalators 4+ N N
4+
N
Ru N N N
N
N
N N
N
N
N N
N
N
N Ru N
N
Kd - equilibrium dissociation constant C - concentration n - binding site size
N
N
N
N
N N
Ru N N N
0.8
0.6
0.4
0.2
0 0.01
The fitting yields values for Kd and n.
N
Kd = 1.89±0.04 nM n = 1.71±0.20
1
Fractional Binding (Θ)
Abstract
0.1
1
10
100
1000
Concentration (nM)
Ru N N N
Quantifying binding kinetics The ΔΔ-P (-[µ-bidppz(phen)4Ru2]4+) has four phenanthroline ancillary ligands (left), while ΔΔ-B (ΔΔ-[μ-bidppz(bipy)4Ru2]4+) has four bipyridine ancillary ligand (right).
The exponential fitting of the constant force measurements yields the total rate of binding.
Large conformational changes of the DNA are needed for these ligands to bind by threading intercalation.
𝑘 = 𝑘𝑜𝑛 + 𝑘𝑜𝑓𝑓
The threading moiety of ΔΔ-P is significantly larger in steric bulk than ΔΔ-B, which suggests that ΔΔP requires larger conformational changes in DNA in order to thread.
The equilibrium dissociation constant can be written as
𝑘𝑜𝑓𝑓 𝐾𝑑 = 𝐶 𝑘𝑜𝑛
Bulk studies have shown the large role steric bulk has on the kinetics and equilibrium behavior of threading intercalators.
0.02
Kon/off (s-1)
Introduction: DNA stretching with optical tweezers
ΔΔ-B held at a constant 30 pN
0.01
Combining the above two relations yields the on and off rates
Constant force measurements of DNA/ΔΔ-B binding 𝑘𝑜𝑛 =
82
In order to facilitate the slow binding, the ligand is introduced to the DNA when it is stretched at constant force.
72
Bare DNA stretch and relaxation
𝑘 𝐾 1+ 𝑑 𝐶
𝑘𝑜𝑓𝑓 =
𝑘 𝐶 1+𝐾 𝑑
0 0
5
10
15
20
25
30
Concentration (nM)
62
When the ligand binds to DNA it will elongate the DNA until an equilibrium binding has been reached (right). Worm Like Chain
82
Dual beam optical tweezers are used to trap a single DNA molecule (top).
52
90 nM 25 nM 12 nM
0.43
42
6.0 nM
32
Freely Jointed Chain
• Overstretching transition
22
• Non-equilibrium strand breaking
12
0.41
2.3 nM 0.39
0.37
0.6 nM
0.35
0.40
0.45
32
Conclusions, future plans, and acknowledgments
0.50
0.55
0.60
0.65
Conclusions
12 2 0.30
0.35
0.40
0.45
0.50
0.55
0.60
Equilibrium elongations at a specific force and varying concentrations are obtained from constant force experiments (left, open circles). These can be modeled as a function of time with a single exponential fitting (left, solid lines).
𝑥 𝑡 = 𝑥0 + ∆𝑥𝑒𝑞 (1 − 𝑒 −𝑘𝑡 )
2 0.30
2.3 nM ΔΔ-B held at a constant 30 pN x(t) →
Extension (nm/bp)
0.45
Extension (nm/bp)
• The elastic regime
Extension as a function of time for ΔΔ-B held at a constant 30 pN
62
Force (pN)
• The entropic regime
42
22
72
In a typical DNA stretching (right), four distinct regimes are observed:
Force (pN)
52
0.35
Extension (nm/bp) 0.33 0
50
100
150
200
Time (s)
250
300
350
𝑥0 - initial DNA elongation ∆𝑥𝑒𝑞 - equilibrium DNA elongation 𝑘 - net relaxation rate
The measured dissociation constant for ΔΔ-B is 6 times smaller than for the similar ΔΔ-P at the same force, implying higher binding affinity to DNA. The kinetics analysis reveals that at 0.6 nM ΔΔ-B binds to DNA 4 times faster than ΔΔ-P, and dissociates from DNA 2 times slower. This implies that the steric bulk of the threading moieties has a key role in the threading of these compounds into DNA.
Future plans Obtain constant force measurements at different forces for ΔΔ-B to determine the zero force behavior of DNA and ΔΔ-B binding. Investigate threading moieties with different chiralities.
Binding Parameters at 30 pN Kd (nM)
n
ΔΔ-B
1.89 ± 0.04
1.71 ± 0.20
ΔΔ-P*
12.0 ± 1.0
3.41 ± 0.20
*Almaqwashi, A. A. et. al. NAR, 2014, 42, 11634-11641
Acknowledgments
The role of the threading moiety in DNA threading intercalation by ruthenium dimer complexes Andrew
1 Clark ,
Thayaparan
1 2 Paramanathan ,
3 Westerlund ,
Fredrik
Per
3 Lincoln ,
1 McCauley ,
Micah J.
Ioulia
4 Rouzina ,
and Mark
1 C.Williams
1Department
of Physics, Northeastern University, Boston, USA. 2 Department of Physics, Bridgewater State University, Bridgewater, USA. 3 Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden. 4Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, USA.
Fitting to McGhee von Hippel Isotherm
Threading intercalation
There is significant interest in ruthenium dimer complexes due to their novel DNA binding properties. The binuclear ruthenium complex ΔΔ-[μ-bidppz(bipy)4Ru2]4+ that we investigate in this study binds to DNA by threading intercalation, in which one of the bulky ruthenium moieties threads through the DNA base pair stack to reach its bound state. The extremely slow dissociation from DNA of this threading intercalator gives it properties needed for chemotherapy drugs, while also making it difficult to study in traditional bulk experiments. In this study, we use optical tweezers in order to quantify the threading kinetics, as well as the equilibrium behavior of this ruthenium dimer complex to understand how changing the ancillary ligands coordinated to ruthenium (threading moiety) affects the process of threading. To observe the slow threading process, the DNA was held at a constant force in the presence of the ligand and the DNA extension was measured as a function of time until the extension reached equilibrium. When the concentration of the ligand is increased during these constant force measurements, the total extension of the DNA will also increase until it becomes saturated at some concentration as all of the ligand binding sites are filled. These measurements of the equilibrium DNA extension allow for a full quantitative description of the binding kinetics and equilibrium behavior of the complex. The preliminary data suggests that the ΔΔ-[μ-bidppz(bipy)4Ru2]4+ complex with its bicyclic bipyridine ligand, shows significantly faster kinetics compared to the ΔΔ-[μbidppz(phen)4Ru2]4+ , complex with a tricyclic phenanthroline ligand counterpart.
The fractional equilibrium binding of ΔΔ-B (right, blue triangles) is estimated by,
During threading intercalation, the molecule must thread a large bulky moiety through dsDNA so that the central planar section can be stacked between the base pairs.
𝑥𝑒𝑞 𝐹, 𝐶 − 𝑥𝑊𝐿𝐶 (𝐹) 𝛩 𝐹, 𝐶 = 𝑥𝑠𝑎𝑡 𝐹 − 𝑥𝑊𝐿𝐶 (𝐹)
In order to dissociate from this state, the ligand has to unthread one of its bulky moieties from the DNA.
This was then fit to the McGhee von Hippel isotherm (right, red).
This results in high DNA binding affinity with extremely slow kinetics.
𝐶 𝑛 ∙ (1 − 𝛩)𝑛 𝛩 𝐾𝑑, 𝑛 = ∙ 𝐾𝑑 (1 − 𝛩 + 𝛩)𝑛−1 𝑛
Paramanathan, T. et. al. JACS 2008, 130, 3752-3753
Semi-rigid ruthenium threading intercalators 4+ N N
4+
N
Ru N N N
N
N
N N
N
N
N N
N
N
N Ru N
N
Kd - equilibrium dissociation constant C - concentration n - binding site size
N
N
N
N
N N
Ru N N N
0.8
0.6
0.4
0.2
0 0.01
The fitting yields values for Kd and n.
N
Kd = 1.89±0.04 nM n = 1.71±0.20
1
Fractional Binding (Θ)
Abstract
0.1
1
10
100
1000
Concentration (nM)
Ru N N N
Quantifying binding kinetics The ΔΔ-P (-[µ-bidppz(phen)4Ru2]4+) has four phenanthroline ancillary ligands (left), while ΔΔ-B (ΔΔ-[μ-bidppz(bipy)4Ru2]4+) has four bipyridine ancillary ligand (right).
The exponential fitting of the constant force measurements yields the total rate of binding.
Large conformational changes of the DNA are needed for these ligands to bind by threading intercalation.
𝑘 = 𝑘𝑜𝑛 + 𝑘𝑜𝑓𝑓
The threading moiety of ΔΔ-P is significantly larger in steric bulk than ΔΔ-B, which suggests that ΔΔP requires larger conformational changes in DNA in order to thread.
The equilibrium dissociation constant can be written as
𝑘𝑜𝑓𝑓 𝐾𝑑 = 𝐶 𝑘𝑜𝑛
Bulk studies have shown the large role steric bulk has on the kinetics and equilibrium behavior of threading intercalators.
0.02
Kon/off (s-1)
Introduction: DNA stretching with optical tweezers
ΔΔ-B held at a constant 30 pN
0.01
Combining the above two relations yields the on and off rates
Constant force measurements of DNA/ΔΔ-B binding 𝑘𝑜𝑛 =
82
In order to facilitate the slow binding, the ligand is introduced to the DNA when it is stretched at constant force.
72
Bare DNA stretch and relaxation
𝑘 𝐾 1+ 𝑑 𝐶
𝑘𝑜𝑓𝑓 =
𝑘 𝐶 1+𝐾 𝑑
0 0
5
10
15
20
25
30
Concentration (nM)
62
When the ligand binds to DNA it will elongate the DNA until an equilibrium binding has been reached (right). Worm Like Chain
82
Dual beam optical tweezers are used to trap a single DNA molecule (top).
52
90 nM 25 nM 12 nM
0.43
42
6.0 nM
32
Freely Jointed Chain
• Overstretching transition
22
• Non-equilibrium strand breaking
12
0.41
2.3 nM 0.39
0.37
0.6 nM
0.35
0.40
0.45
32
Conclusions, future plans, and acknowledgments
0.50
0.55
0.60
0.65
Conclusions
12 2 0.30
0.35
0.40
0.45
0.50
0.55
0.60
Equilibrium elongations at a specific force and varying concentrations are obtained from constant force experiments (left, open circles). These can be modeled as a function of time with a single exponential fitting (left, solid lines).
𝑥 𝑡 = 𝑥0 + ∆𝑥𝑒𝑞 (1 − 𝑒 −𝑘𝑡 )
2 0.30
2.3 nM ΔΔ-B held at a constant 30 pN x(t) →
Extension (nm/bp)
0.45
Extension (nm/bp)
• The elastic regime
Extension as a function of time for ΔΔ-B held at a constant 30 pN
62
Force (pN)
• The entropic regime
42
22
72
In a typical DNA stretching (right), four distinct regimes are observed:
Force (pN)
52
0.35
Extension (nm/bp) 0.33 0
50
100
150
200
Time (s)
250
300
350
𝑥0 - initial DNA elongation ∆𝑥𝑒𝑞 - equilibrium DNA elongation 𝑘 - net relaxation rate
The measured dissociation constant for ΔΔ-B is 6 times smaller than for the similar ΔΔ-P at the same force, implying higher binding affinity to DNA. The kinetics analysis reveals that at 0.6 nM ΔΔ-B binds to DNA 4 times faster than ΔΔ-P, and dissociates from DNA 2 times slower. This implies that the steric bulk of the threading moieties has a key role in the threading of these compounds into DNA.
Future plans Obtain constant force measurements at different forces for ΔΔ-B to determine the zero force behavior of DNA and ΔΔ-B binding. Investigate threading moieties with different chiralities.
Binding Parameters at 30 pN Kd (nM)
n
ΔΔ-B
1.89 ± 0.04
1.71 ± 0.20
ΔΔ-P*
12.0 ± 1.0
3.41 ± 0.20
*Almaqwashi, A. A. et. al. NAR, 2014, 42, 11634-11641
Acknowledgments
