A R T I C L E S
Brown et al.
3
3
1
2
′ (X ) AFB-N7-dGuo) was separated from 5′-d(CTATGATTCA)-
′ and 5′-d(AATCATA)-3′ using HPLC (Gemini C-18 250 mm ×
0 mm column, Phenomenex, Inc., Torrance, CA) at a flow rate of
at NOE mixing times of 80, 150, 200, and 250 ms were conducted
using isotropic correlation times of 2, 3, 4, and 5 ns; analysis of
the resulting distances produced the distance restraints used in rMD
calculations. Torsion angle restraints were determined from 31P- H
heterocorrelation, DQF-COSY, and NOESY spectra. Phosphodiester
1
3
mL/min, with a linear 25 min gradient of 6-30% CH CN in 0.1
M ammonium formate (pH 8.0). The eluant was monitored by UV
absorbance at 260 and 360 nm. The yield of 5′-d(CTATXATTCA)-
9
6-98
backbone restraints
except that the backbone torsion angles at X and C were not
restrained. The ꢂ to ꢂ deoxyribose torsion angles were determined
were applied to the R, ꢀ, γ, ν
0
-ν
4
angles
5
16
3
′ (X ) AFB-N7-dGuo) was 90%. The 5′-d(CTATXATTCA)-3′
(
X ) AFB-N7-dGuo) was suspended in 500 mL of 100 mM
Na CO at 37 °C (pH 10). The AFB-R-FAPY and AFB-ꢀ-FAPY
modified oligodeoxynucleotides were separated by HPLC as
described above and collected in a saturated Na HPO solution.
0
4
9
9
2
3
by pseudorotation analysis. For the N-type conformation, the
phase angle F was restrained between 0° and 205°, and for the
S-type conformation, the phase angle F was restrained between 110°
and 205°.
2
4
The AFB-R-FAPY modified 5′-d(CTATXATTCA)-3′ oligodeoxy-
nucleotide was desalted using a C-18 Sep-Pak cartridge (Waters
Corp, Milford, MA) before annealing with 5′-d(TGAATCATAG)-
Structural Refinement. The refinement used a simulated an-
4
4-47
nealing rMD protocol involving established methods,
employing generalized Born solvation
and
1
00,101
102
3
′ in a 1:1 molar ratio in 0.2 M sodium phosphate, 0.1 M NaCl, 10
with the AMBER
mM NaN
3 2
, and 50 µM Na EDTA (pH 8.7) to yield a duplex
force field. A 15 Å cutoff for nonbonded interactions was used,
and bond lengths involving hydrogen were fixed using the SHAKE
concentration of 1 mM. For the tetramer 5′-d(CTGA)-3′, the
adduction reaction utilized a 5:1 epoxide/oligodeoxynucleotide ratio
that provided a 40% yield of adducted tetramer at strand concentra-
tions >1 mM. Its purification, conversion to the FAPY adduct, and
isolation of the oligodeoxynucleotide containing the AFB-R-FAPY
adduct followed the protocols described above. All samples were
stored at 5 °C except during spectroscopic measurements.
Thermal Melting. UV thermal melting analyses were conducted
in 1 mL of 10 mM sodium phosphate, 0.5 M NaCl, and 5 mM
Na
of all samples was maintained at 0.1 A260 unit under denaturing
conditions. The T values were determined from first derivatives
1
03
algorithm.
The emergent structures from 20 calculations with
different starting velocities were analyzed as to pairwise rmsd
deviations. This provided a measure of the precision of the
calculations. Complete relaxation matrix analysis was performed
5
2,53
using the program CORMA.
This provided a measure of the
accuracy of the calculations. The output from the simulated
annealing calculations was also validated with respect to restraint
violations. Graphical analysis was performed with InsightII (Ac-
5
4
2
EDTA (pH 7.0) and monitored at 260 nm. The concentration
celrys, San Diego, CA) and CHIMERA.
Isothermal rMD Calculations in Explicit Solvent. The starting
structure was a representative potential energy minimized structure
from the ensemble of refined structures for the duplex containing
the AFB-R-FAPY adduct emergent from the simulated annealing
m
of the absorbance versus temperature curves. The experiments
temperature range was 5-80 °C.
1
44
+
NMR Spectroscopy. Spectra were recorded at H frequencies
calculations. The DNA was neutralized with Na ions and placed
in a truncated octahedral TIP3P water box with periodic boundaries
at a distance of 8 Å from the solute. Sodium ions were constrained
to be proximate to sequential backbone phosphates using restraints
having a lower bound of 3.0 Å and an upper bound of 8.0 Å. The
solvated system was energy minimized for 500 steps of steepest
descents followed by 500 steps of conjugate gradients at constant
volume with the solute held fixed. Next, the system was subjected
to potential energy minimization at a constant volume for 2500
steps with no positional restraints. The system was subsequently
heated to 300 K over 100 ps. The experimental distance and torsion
angle restraints and empirical restraints (Vide supra) were increased
of 500, 600, and 800 MHz. To examine nonexchangeable protons,
samples were suspended in D
protons, samples were suspended in 9:1 H
XWINNMR and TOPSPIN (Bruker, Inc., Billerica, MA) were used
for data processing. Resonance assignment and peak integration
2
O. For observation of exchangeable
O/D O. The programs
2
2
85
were performed using the program SPARKY. COSY spectra were
8
6
conducted in double quantum filtered (DQF) and magnitude
modes. A series of experiments were collected at NOE mixing times
of 80, 150, 200, and 250 ms; the NOESY spectra for the single
strand tetramer samples were collected at mixing times of 250 and
8
7,88
3
00 ms. NOESY
experiments used for the assignment of
89
exchangeable protons used the watergate pulse sequence. Water
suppression for heteronuclear H- P correlation experiments was
achieved with the Dante pulse sequence. Pulses were optimized
linearly during the heating. Isothermal rMD calculations were
1
31
90
102
performed for 5 ns at 300 K using the AMBER
force field,
9
1
restrained by experimental distance and torsion angle restraints and
empirical restraints. The temperature was maintained using the
3
for 12 Hz JPH coupling constants.
1
04,105
-1
NMR Restraints. NOE intensities were determined from
Langevin thermostat
with a collision frequency of 1 ps .
8
5
integration of cross-peak volumes using the program SPARKY.
Electrostatic interactions were treated with the particle mesh Ewald
1
06
Experimental intensities were combined with those generated from
complete relaxation matrix analysis of a model structure, using
52,53
(PME) method. A 15 Å cutoff for nonbonded interactions was
44
established methodologies, to produce a hybrid intensity matrix.
94,95
(95) Spielmann, H. P.; Dwyer, T. J.; Hearst, J. E.; Wemmer, D. E.
9
2,93
Biochemistry 1995, 34, 12937–12953.
96) Mujeeb, A.; Kerwin, S. M.; Kenyon, G. L.; James, T. L. Biochemistry
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using the RANDMARDI
(
function, was used to refine the hybrid intensity matrix. Calculations
1
993, 32, 13419–13431.
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97) Wijmenga, S. S.; Mooren, M. M. W.; Hilbers, C. W. In NMR of
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1
6106 J. AM. CHEM. SOC. 9 VOL. 131, NO. 44, 2009