accelerating voltage of 25 000 V. Acquisition was between 4000–
15000 Da with 100 shots/spectrum.
GWL-78 (1) and anthramycin (2) were graphically aligned in the
minor groove of the DNA using the ‘Xleap’ program so that
the A-ring of the PBD was oriented towards the 3¢-end of the
helix, and the N10-nitrogen of the 7-membered ring was in close
proximity to the NH2 of guanine. Use was made of ‘parm99¢
and the general Amber force field parameters ‘gaff’. Subsequent
minimization steps were performed with the DNA and unbound
ligand using ‘Sander’ in such a way that the DNA was initially
restrained with a high force constant, thus allowing the ligand to
adjust to the DNA environment. Further minimization steps were
then performed while gradually reducing the restraints to zero. The
generalized Born/surface area (GB/SA) implicit solvent model
was used with monovalent electrostatic ion screening simulated
with SALTCON set to 0.2 M. A long range non-bonded cut-off
EI-MS. EI-MS spectra were acquired on a Micromass Q-
TOF Global Tandem Mass Spectrometer (Waters, Manchester,
UK) fitted with a NanoSpray ion source. Negative mode was
used for data acquisition, and the instrument was calibrated
with ions produced from a standard solution of taurocholic acid
(10 pmole mL-1) in acetonitrile. The HPLC fractions collected
were lyophilised (SpeedVac, Thermo Electron, UK) and mixed
with a 1 : 1 v/v mixture of 40% acetonitrile–water and 20 mM
triethylamine–water (TEA, Fischer Scientific, UK) which was
also used as the electrospray solvent. 3–5 mL of sample was
loaded into a metal-coated borosilicate electrospray needle with
an internal diameter of 0.7 mm and a spray orifice of 1–10 mm
(NanoES spray capillaries, Proxeon Biosystems, UK), and this
was positioned ~10 mm from the sample cone to provide a flow
rate of ~20 nL min-1. Nitrogen was used as the API gas, and the
capillary, cone and RF Lens 1 voltages were set to 1.8–2.0 kV,
~ 35 V and 50 V, respectively, to ensure minimum fragmentation
of the ligand/DNA adducts. The collision voltage was set to 5 V
and the MCP voltage to 2200 V. Spectra were acquired over the
100–1500 m/z range.
˚
of 100 (A) was used. Final models were visualized with the VMD
and PyMol24 programs.
Acknowledgements
Dr Emma Sharp is thanked for her help with preparation of
the manuscript. Spirogen Ltd is acknowledged for supplying a
sample of GWL-78 (1) and for providing the funding for the
oligonucleotides. Drs Alex Drake and Tam T.T. Bui of Kings
College London are thanked for carrying out the CD spectroscopic
measurements.
Circular Dichroism and Thermal Denaturation Studies. The
UV
& CD spectra of the oligonucleotides and oligonu-
cleotide/ligand complexes were acquired on a Chirascan spec-
trometer (Applied Photophysics Ltd, Leatherhead, UK). The UV
absorbance and CD spectra were measured between 500–200 nm
in a strain-free rectangular 10 mm cell. The instrument was
flushed continuously with pure evaporated nitrogen throughout
the experiments. Spectra were recorded using a 0.5 nm step size, a
1.5 s time-per-point and a spectral bandwidth of 1 nm. Addition
of ligand to the oligonucleotide solutions was carried out while
maintaining a constant concentration of DNA. All spectra were
acquired at room temperature and the buffer baseline corrected.
All CD spectra were smoothed using the Savisky-Golay method,
and a window factor of 4–12 was used for a better presentation.
For thermal denaturation experiments, the CD spectra were
first recorded at room temperature (20 ◦C), then at the highest
temperature (90 ◦C), and then again after cooling to 20 ◦C. Melting
profiles were recorded during both the heating and cooling phases.
The instrument was equipped with a Quantum (NorthWest, USA)
TC125 Peltier unit set to change temperature from 20→90 ◦C at a
rate of 3 ◦C min-1, with a 5 ◦C step-size and a 0.2 ◦C tolerance. The
References
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◦
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1640 | Org. Biomol. Chem., 2011, 9, 1632–1641
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The Royal Society of Chemistry 2011
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