V. Satam et al. / Bioorg. Med. Chem. 20 (2012) 693–701
701
Sciences). DNA hairpin constructs labeled with biotin at the 50 end
were immobilized onto a streptavidin-coated sensor chip (sensor
chip SA) as previously reported.20a,b,22 The 50-biotin-labeled DNA
hairpins were immobilized on three of the four flow cells. The
fourth was left blank and used as a control. All SPR experiments
were performed at 25 °C and used 0.01 M CCA as the running buf-
fer. The amount of DNA immobilized was approximately 400 re-
sponse units (RU). This was achieved by continuously injecting
2. (a) Chou, C. J.; Farkas, M. E.; Tsai, S. M.; Alvarez, D.; Dervan, P. B.; Gottesfeld, J.
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ꢃ20
lL of an approximately 50 nM DNA solution at a rate of
L/min onto the sensor chip surface until a relative response of
2
l
400 units was reached. Binding data were obtained by injecting
known concentrations and were analyzed with one or two site
binding models as previously described.20a,b,22
r ¼ ðK1 ꢁ Cfree þ 2 ꢁ K1 ꢁ K2 ꢁ C2 Þ=ð1 þ K1 ꢁ Cfree þ K1 ꢁ K2
free
ꢁ C2free
Þ
ð1Þ
where, r represents the moles of bound compound per mole of DNA
hairpin duplex, K1 and K2 are macroscopic binding constants, and
Cfree is the free compound concentration in equilibrium with the
complex.
4.2.6. Isothermal titration microcalorimetry (ITC)
ITC analysis was performed using a VP-ITC microcalorimeter
(MicroCal). Compound 3 was dissolved in 0.01 M cacodylic acid
(CCA) buffer and the instrument equilibrated to 25 °C. ITC experi-
ments were conducted using the ‘model-free’ method as previously
reported.23 {insert B Chaires ref here (Biochem 2000, 39, 8439–
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Lett. 1991, 1, 595.
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M.; Foister, S.; Heckel, A.; Dervan, P. B. Nucleic Acids Res. 2004, 32, 2802; (c)
Best, T. P.; Edelson, B. S.; Nickols, N. G.; Dervan, P. B. Proc. Natl. Acad. Sci. 2003,
100, 12063.
14. Lown, J. W.; Krowicki, K.; Bhat, U. G.; Skorobogaty, A.; Ward, B.; Dabrowiak, J. C.
Biochemistry 1986, 25, 7408.
15. Satz, A. L.; Bruice, T. C. Acc. Chem. Res. 2002, 35, 86.
16. Babu, B.; Liu, Y.; Plaunt, A.; Riddering, C.; Ogilvie, R.; Westrate, L.; Davis, R.;
Ferguson, A.; Mackay, H.; Rice, T.; Chavda, S.; Wilson, D.; Lin, S.; Kiakos, K.;
Hartley, J. A.; Lee, M. Biochem. Biophys. Res. Commun. 2011, 404, 848.
17. Lee, M.; Rhodes, A.; Wyatt, M. D.; Forrow, S.; Hartley, J. A. Biochemistry 1993,
32, 4237.
18. Chavda, S.; Liu, Y.; Babu, B.; Davis, R.; Sielaff, A.; Ruprich, J.; Westrate, L.;
Tronrud, C.; Ferguson, A.; Franks, A.; Tzou, S.; Adkins, C.; Rice, T.; Mackay, H.;
Kluza, J.; Tahir, S. A.; Lin, S.; Kiakos, K.; Bruce, C. D.; Wilson, D. W.; Hartley, J. A.;
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8447)}. After an initial delay of 300 s, compound 3 (80
trated, via 30 injections (10 L for 20 s, repeated every 300 s), into
30 M DNA (0.01 M CCA). The data were imported to Origin 7.0,
lM) was ti-
l
l
which was used to integrate the area under the curve as a function
of time. In order to normalize for nonspecific heat components,
including heats of dilution, nonspecific heat component integrations
were subtracted from the reaction integrations.5 A linear fit of the
normalized
DH (y-axis) values versus mol ratio (x-axis) was done
using KaleidaGraph 4.0. Extrapolation back to 0 mol ratio of this plot
yields the enthalpy (DH) of the reaction. The DNA used in this exper-
iment was 50-ACGCGT-30, 50-AAATTT-30, and 50-ACCGGT-30.
19. Westrate, L.; Mackay, H.; Brown, T.; Nguyen, B.; Kluza, J.; Wilson, D. W.; Lee,
M.; Hartley, J. A. Biochemistry 2009, 48, 5679.
Acknowledgments
20. (a) Liu, Y.; Wilson, W. D. Methods Mol. Biol. 2010, 613, 1; (b) Lacy, E. R.; Minh Le,
N.; Price, C. A.; Lee, M.; Wilson, W. D. J. Am. Chem. Soc. 2002, 124, 2153; (c) Le
Minh, N.; Sielaff, A.; Cooper, A. J.; Mackay, H.; Brown, T.; Kotecha, M.; O’Hare,
C.; Hochauser, D.; Lee, M.; Hartley, J. A. Bioorg. Med. Chem. Lett. 2006, 16, 6161;
(d) Buchmueller, K. L.; Staples, A. M.; Uthe, P. B.; Howard, C. M.; Pacheco, K. A.
O.; Cox, K. K.; Henry, J. A.; Bailey, S. L.; Horick, S. M.; Nguyen, B.; Wilson, D. W.;
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742.
The authors thank the NSF [CHE 0809162, CHE 0550992, and
CHE 0922623 (NMR)], Cancer Research UK (C2259/A9994 to JAH)
and the Georgia Research Alliance for their generous support.
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
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