Chemistry Reference
In-Depth Information
dilution experiments were unsuccessful. No significant upfield shifts of the aniline
NH signals were observed across a broad concentration range (100 mM to 1 mM),
suggesting a lower limit of 9
10
7
M
21
for the association constant of duplex 3†4
and assuming a 10% dissociation at 1 mM. Isothermal titration calorimetry (ITC)
could only give an estimated value of
10
9
M
21
. The high stability of 3†4 even
allowed its detection by straight-phase thin layer chromatography (TLC) under rather
polar conditions [silica gel plate, 10% dimethylformamide (DMF) in chloroform].
The
1
H-NMR spectra of mixtures of 3 and 4 in stoichiometries other than 1:1 revealed
separate sets of signals corresponding to both duplex 3†4 and the uncomplexed strands.
This indicated that exchange between the assembled and the uncomplexed states
was very slow on the timescale of NMR spectroscopy, which suggested high kinetic
stability for the duplex. Although efforts to crystallize 3†4 have not succeeded, the
presence of 3†4 in solution was unequivocally demonstrated by numerous interstrand
NOEs in the NOESY spectrum of this compound (Fig. 9.3a).
Analysis of the pyrene-labeled homoduplex 5†5 (Fig. 9.3b) by NMR, mass
spectrometry, and TLC suggested that 5†5 had a stability similar to that of 3†4.
NOESY spectra revealed cross-strand NOEs consistent with the formation of the
self-dimer 5†5 (Zeng et al. 2003). Based on a fluorescence method described in
the literature (Sontjens et al. 2000), the dimerization constant of the pyrene-labeled
duplex 5†5 was found to be (6.77+4.12)
10
9
M
21
. The studies on duplexes
3†4 and 5†5 clearly demonstrated that the stabilities of our duplexes are indeed
only determined by the number of intermolecular H bonds, and both hetero- and
homoduplexes can be easily designed and constructed.
Thus, the stability is enhanced by more than 5 orders of magnitude with two
additional H bonds (i.e., the quadruply vs. 6-H bonded duplexes), which implies the
presence of positive cooperativity in this H bonded system. Comparing the stabilities
of doubly (K
a
¼ 25 M
21
, -0.9 kcal/mol for each H bond) and quadruply (dimers of
1 and 2,10
4
M
21
, K
a
, 10
5
M
21
, -1.4 to -1.7 kcal mol
21
for each H bond) H
bonded duplexes with that of 3†4 (1
10
9
M
21
, 2.0 kcal/mol for each H bond) indi-
cates that the increase in stabilities is not just due to the additive effect from increased
numbers of hydrogen bonds. Instead, these data clearly demonstrate that the self-
assembly of 3†4 is highly cooperative: after the initial association of the two
strands, which may involve one or, at most, two hydrogen bonds and is entropically
unfavorable, the subsequent formation of H bonds during the growth of the duplex is
enhanced by multiple, enthalpically favorable interactions. This type of cooperativity
is one of the most prominent features of the self-assembly of DNA and lies at the heart
of many self-assembling and self-organizing systems (Lindsey 1991).
9.5. PROBING SEQUENCE SPECIFICITY
Sequence-specific pairing of DNA and RNA strands is essential for the storage,
transmission, and expression of genetic information, which forms the basis for tech-
niques such as polymerase chain reaction, hybridization techniques, and DNA chip
arrays. Having demonstrated the sequence-independent nature in the stabilities of
