Chemistry Reference
In-Depth Information
ions to compete with Na
+
ions for coordination within the G-quadruplex d[(G
4
T
4
G
4
)
2
]
has been verifi ed by solution-state
1
H NMR.
128
Differences in resonance peak line
widths for the three Tl
+
resonances suggests that ions move from one cation binding
site to the other. The central peak was narrower, which was attributed to the slower
exchange of the Tl
+
ion at the middle coordination site. The outside Tl
+
ions exchange
with bulk solution more freely. The lifetime of Tl
+
ions bound within the G-
quadruplex was estimated to be at least 3 m s.
127
One of the fi rst NMR studies to determine the number of cations bound within
a G-quadruplex in the solution state was performed using the sequence d(G
3
T
4
G
3
).
168
In the presence of either NaCl or KCl, d(G
3
T
4
G
3
) forms a bimolecular fold-back
structure (Figure 3.6d).
238,240
1
H NMR spectroscopy was used to follow the competi-
tion between Na
+
and K
+
ions for coordination sites within the G-quadruplex.
168
Changes in
1
H NMR spectra during these titration experiments indicated a gradual
transition of d[(G
3
T
4
G
3
)
2
] from the G-quadruplex structure observed in the pres-
ence of only NaCl to the G-quadruplex structure observed in presence of only KCl.
Although the Na
+
and K
+
forms of d[(G
3
T
4
G
3
)
2
] have the same molecular folds,
there are relatively small structural differences between the two forms that are
manifested by changes in
1
H chemical shifts. No separate or additional
1
H reso-
nances were observed in samples of d[(G
3
T
4
G
3
)
2
] that contained various mixtures
of NaCl and KCl, which demonstrated that the exchange rate of Na
+
and K
+
forms
of this quadruplex at 25 °C is fast on the NMR timescale (ca.
10 ms).
1
H chemical
shift changes observed over the course of KCl titration into a sample of d[(G
3
T
4
G
3
)
2
]
were fi t perfectly by a model in which two Na
+
ions are replaced by two K
+
ions
within the G-quadruplex. Furthermore, quantitative analysis of chemical shift
changes during the same titration experiments
168
gave important insights regarding
the thermodynamic basis for the preferential coordination of K
+
over Na
+
by
G - quadruplexes (see below).
d[G
4
(T
4
G
4
)
3
] has been folded into a monomolecular G-quadruplex in the pres-
ence of
15
NH
4
+
ions (Figure 3.2 c).
164,255
Topology of the folded structure is equivalent
to the solution-state structure in the presence of Na
+
ions determined earlier in the
laboratories of Patel
256
and Feigon.
257
The
15
NH
4
+
ion binding sites within the
d[G
4
(T
4
G
4
)
3
] G - quadruplex were identifi ed and assigned with the use of 2D
1
H -
15
N
HSQC and NOESY spectra.
164
The 3D structure of this monomolecular G-
quadruplex consists of four G-quartets with alternating parallel and antiparallel
strands (Figure 3.2c).
256,257
The conformation of guanines along the strands alternates
between
syn
and
anti
. One of the T
4
loops spans diagonally across the outer G-
quartet, while the two T
4
loops are in an edge-wise conformation. Three
15
NH
4
+
ion
binding sites were established within the interior of d[G
4
(T
4
G
4
)
3
].
164
There was no
indication of
15
NH
4
+
ions residing at the loop binding sites. The volumes of HSQC
cross-peaks corresponding to the three binding sites were not the same. The outer
site (named O
1
), which is closer to the outer G-quartet spanned by the diagonal
loop showed the highest occupancy. The inner binding site, I, and the other outer
site, O
2
, demonstrated comparable occupancies at 130 mM
15
NH
4
Cl.
164
Quantitative
analysis afforded the equilibrium binding constants of 93, 31 and 46 M
− 1
for O
1
, I
and O
2
binding sites.
255
The binding site I which is in the core of d[G
4
(T
4
G
4
)
3
] exhibits
the lowest affi nity for
15
NH
4
+
ions. The binding site O
1
exhibits a three times higher
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