TY - JOUR
T1 - Binding of netropsin and 4,6-diamidino-2-phenylindole to an A2T2 DNA hairpin
T2 - A comparison of biophysical techniques
AU - Freyer, Matthew W.
AU - Buscaglia, Robert
AU - Nguyen, Binh
AU - David Wilson, W.
AU - Lewis, Edwin A.
N1 - Funding Information:
This work was funded in part by Arizona Biomedical Research Commission (ABRC) award numbers 05-007 and 05-002 A (to E.A. Lewis) and by National Institutes of Health (NIH) Grant AI064200 (to W.D. Wilson). We thank Brad Chaires (University of Louisville) for helping to initiate this research project and for many helpful discussions.
PY - 2006/8/15
Y1 - 2006/8/15
N2 - Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and biosensor-surface plasmon resonance (SPR) are evaluated for their accuracy in determining equilibrium constants, ease of use, and range of application. Systems chosen for comparison of the three techniques were the formation of complexes between two minor groove binding compounds, netropsin and 4,6-diamidino-2-phenylindole (DAPI), and a DNA hairpin having the sequence 5′-d(CGAATTCGTCTCCGAATTCG)-3′. These systems were chosen for their structural differences, simplicity (1:1 binding), and binding affinity in the range of interest (K ∼ 108 M-1). The binding affinities determined from all three techniques were in excellent agreement; for example, netropsin/DNA formation constants were determined to be K = 1.7 × 108 M-1 (ITC), K = 2.4 × 108 M-1 (DSC), and K = 2.9 × 108 M-1 (SPR). DSC and SPR techniques have an advantage over ITC in studies of ligands that bind with affinities greater than 108 M-1. The ITC technique has the advantage of determining a full set of thermodynamic parameters, including ΔH, TΔS, and ΔCp in addition to ΔG (or K). The ITC data revealed complex binding behavior in these minor groove binding systems not detected in the other methods. All three techniques provide accurate estimates of binding affinity, and each has unique benefits for drug binding studies.
AB - Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and biosensor-surface plasmon resonance (SPR) are evaluated for their accuracy in determining equilibrium constants, ease of use, and range of application. Systems chosen for comparison of the three techniques were the formation of complexes between two minor groove binding compounds, netropsin and 4,6-diamidino-2-phenylindole (DAPI), and a DNA hairpin having the sequence 5′-d(CGAATTCGTCTCCGAATTCG)-3′. These systems were chosen for their structural differences, simplicity (1:1 binding), and binding affinity in the range of interest (K ∼ 108 M-1). The binding affinities determined from all three techniques were in excellent agreement; for example, netropsin/DNA formation constants were determined to be K = 1.7 × 108 M-1 (ITC), K = 2.4 × 108 M-1 (DSC), and K = 2.9 × 108 M-1 (SPR). DSC and SPR techniques have an advantage over ITC in studies of ligands that bind with affinities greater than 108 M-1. The ITC technique has the advantage of determining a full set of thermodynamic parameters, including ΔH, TΔS, and ΔCp in addition to ΔG (or K). The ITC data revealed complex binding behavior in these minor groove binding systems not detected in the other methods. All three techniques provide accurate estimates of binding affinity, and each has unique benefits for drug binding studies.
KW - Biosensor-surface plasmon resonance
KW - DAPI
KW - DSC
KW - Differential scanning calorimetry
KW - Hairpin DNA
KW - ITC
KW - Isothermal titration calorimetry
KW - Minor groove binding affinity
KW - Netropsin
KW - SPR
KW - Thermodynamics
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U2 - 10.1016/j.ab.2006.04.049
DO - 10.1016/j.ab.2006.04.049
M3 - Article
C2 - 16828700
AN - SCOPUS:33746312998
SN - 0003-2697
VL - 355
SP - 259
EP - 266
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -