Cooperative melting in caged dimers with only two DNA duplexes

Article abstract of DOI:10.1021/ja107232x

Small molecule-DNA hybrids with only two parallel DNA duplexes (rSMDH ₂) displayed sharper melting profiles compared to unmodified DNA duplexes, consistent with predictions from neighboring-duplex theory. Using adjusted thermodynamic parameters obtained from a coarse-grain dynamic simulation, the experimental data fit well to an analytical model.

Full text of DOI:10.1021/ja107232x

Published on Web 11/12/2010
Cooperative Melting in Caged Dimers with Only Two DNA Duplexes
Ibrahim Eryazici,† Tatiana R. Prytkova,‡ George C. Schatz,*,† and SonBinh T. Nguyen*,†
Department of Chemistry, Northwestern UniVersity, 2145 Sheridan Road, EVanston, Illinois 60208-3113
Received August 11, 2010; E-mail: schatz@chem.northwestern.edu; stn@northwestern.edu
midite chemistry (Figure 1). As an improvement from our previous
Abstract: Small molecule-DNA hybrids with only two parallel
DNA duplexes (rSMDH2) displayed sharper melting profiles
compared to unmodified DNA duplexes, consistent with predic-
tions from neighboring-duplex theory. Using adjusted thermody-
namic parameters obtained from a coarse-grain dynamic simu-
lation, the experimental data fit well to an analytical model.
synthesis,18 we added an extra ethylene glycol (CH2CH2O) linker
between the core and the DNA strand; this makes the products more
stable during the DMT-on reversed-phase HPLC purification. To
evaluate the effect of the core C on melting behavior, we also
prepared analogous SMDH2’s where the arms are simply linked
together via a 6-base pair ss-DNA TTTTTT sequence (T6 spacer;
i.e., 3′-A-5′-T6-3′-B-5′ or A-T6-B for short).
DNA-hybrid materials have been explored in a wide range of
research areas such as sensors and actuators,1,2 molecular electron-
ics,3 template synthesis,4,5 and computing.6-11 In diagnostics, DNA-
linked gold nanoparticle (GNP)12 and comb-like polymer13 aggre-
gates possess a unique switch-like melting property, which has been
utilized to distinguish single-nucleotide polymorphisms (SNPs). This
switch-like melting phenomenon can be explained by a “neighbor-
ing-duplex/effective-concentration” (ND/EC) model,14 which pre-
dicts an enhanced melting temperature (Tm) and sharp melting due
to two effects: (1) entropic constraints associated with the melting
of structures in which the tethered DNAs have a higher effective
concentration than when they are free in solution, and (2) an
increase in the effective ion concentration between neighboring
duplexes that leads to the higher overall stability of fully hybridized
structures and cooperative thermal melting.15 This model, which
has been verified using coarse-grain molecular dynamics (CGMD)
simulations,14 predicts that stabilization can be observed with as
few as 2-3 parallel DNA duplexes, giving rise to sharper melting
transitions in DNA-linked nanoparticle12 and comb-like polymer13
aggregates than that observed for the double-stranded DNA
duplex.16,17
We recently reported the synthesis and cooperative melting
behavior of a small molecule-DNA hybrid containing only three
DNA duplexes (SMDH3) around a rigid (r) small-molecule core
(C).18 Herein, we report the cooperative melting of SMDH2,
possessing only two closely associated double-stranded DNAs, with
or without a rigid core. Using adjusted thermodynamic parameters
obtained from coarse-grain molecular dynamics simulations, the
experimental data fit well to an analytical model where the melting
of these dimeric duplexes is considered as two distinct steps where
most of the entropy is released in the second step.
rSMDH2 3 and 3′ (Figure 1, 3 ) 3′-A-5′-T3CT3-3′-B-5′, 3′ )
3′-A′-5′-T3CT3-3′-B′-5′) containing two asymmetric strands (one
of the strands is the reverse sequence of the other) were designed
to facilitate only the formation of cage-like dimers and avoid face-
to-face bulge-containing DNA structures (Figure S3 in the Sup-
porting Information (SI)). Synthesis of asymmetric rSMDH2’s was
achieved by adding the phosphoramidite core 119 to the initial DNA
arm grown from the surface of a controlled porosity glass bead
(CPG), followed by synthesis of the second arm via 3′-phosphora-
Figure 1. Synthesis of rSMDH2-A-T3CT3-B and its dimeric structures 4-6
(dimeric cages 5 and 6 are formed using rSMDH2, an unmodified DNA
with T6 linker).
Hybridization of rSMDH2’s was performed by combining
equimolar amounts of two complementarily functionalized
rSMDH2’s in phosphate buffer at 25 °C, annealing the hybridized
mixture at 85 °C for 10 min, and allowing it to cool to room
temperature over 4 h. The melting profile of the hybridized mixture
† Northwestern University.
‡ Current Address: Schmid College of Science, Chapman University, One
University Drive, Orange California 92866.
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10.1021/ja107232x  2010 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Melting Data for 15-bp DNA Duplexes (A:A′), A:A′-T6-B′:B,
Caged- (4, 5, 6), and Hairpin-like (8) Dimers Formed by
Unmodified DNA and rSMDH2 Hybrids
was obtained by heating the samples from 20 to 70 °C at a rate of
1 °C per minute while monitoring the increase in UV-vis
absorbance at 260 nm at 0.1 °C intervals. For comparison, we also
formed cage-like structures 5 and 6, from rSMDH2 3 with the
complementary A′-T6-B′, and from A-T6-B:A′-T6-B′, respectively
(Figure 1).
hybridization
mixture
[NaCl]
(mM)a
Tm
(°C)
fwhm
(°C)
Nc
((0.1)
entry
1
2
3
4
5
6
7
8
A:A′b
75
150
300
300
75
150
300
75
150
300
75
150
300
75
150
300
300
75
150
300
35.6 ( 0.4
40.9 ( 0.2
45.3 ( 0.4
45.8
10.3 ( 0.4
10.6 ( 0.1
10.8 ( 0.3
10.5
-
A:A′b
A:A′b
A:A′
-
Similar to the previously reported rSMDH3 experiment,18 non-
denaturing PAGE-gel analyses of the hybridized 0.38-2 µM
solutions containing caged dimers 4 and 6 (Figure 2; see also
Figures S8 and S9 in the SI) displayed sharp bands. However, at
higher concentrations (4-16 µM), solutions of these caged dimers
show new bands and smearing, corresponding to higher cyclic and
oligomeric structures, respectively.
-
-
b c
,
A:A′-T6-B′:Bd
36.4 ( 0.1
41.0 ( 0.3
46.5 ( 0.2
37.5 ( 0.2
43.5 ( 0.4
49.1 ( 0.3
36.9 ( 0.1
43.5 ( 0.1
47.9 ( 0.1
36.5 ( 0.1
42.2 ( 0.1
48.5 ( 0.1
48.8
10.2 ( 0.1
10.3 ( 0.1
10.3 ( 0.3
6.3 ( 0.2
5.9 ( 0.1
7.5 ( 0.1
5.9 ( 0.1
5.8 ( 0.1
6.2 ( 0.1
6.0 ( 0.1
6.1 ( 0.1
5.2 ( 0.2
4.7
1.0
1.0
1.0
1.7
1.8
1.6
2.0
2.1
1.8
1.8
2.0
1.8
-
A:A′-T6-B′:Bd
A:A′-T6-B′:Bd
4b
4b
4b
5b
5b
5b
6b
6b
6b
9
10
11
12
13
14
15
16
17
18
19
20
b c
,
6
8
8
b e
,
35.4 ( 0.2
41.8 ( 0.1
46.6 ( 0.1
8.9 ( 0.2
9.1 ( 0.3
8.9 ( 0.1
1.1
1.2
1.3
b e
,
8b e
,
a NaCl concentration in a 10 mM PBS buffer. b 0.76 µM total
[DNA]. c Theoretical value. d 2.00 µM total [DNA]. e Hairpin-like dimer
8 (3:7).
Figure 2. Nondenaturing PAGE-gel image (10%) of DNA duplexes and
hybrids. From left to right: lane 1 ) HL5 DNA ladder, lane 2 ) A:A′ (2 µM),
lanes 3-6 ) rSMDH2-A-T3CT3-B:rSMDH2-A′-T3CT3-B′ (0.38 to 8 µM), lane
7 ) A:A′-T6-B′:B (2.0 µM), lane 8 ) A:rSMDH2-A′-T3CT3-B′:B (2.0 µM).
The neighboring-duplex/effective-concentration model15 predicts
that when an aggregate possesses two or more parallel DNA
duplexes in close proximity (25-40 Å),17 its melting point (Tm)
increases and its melting profile sharpens. Consistent with this
conjecture, the rSMDH2 dimer 4 (3:3′) displayed a sharper melting
profile (full width at half-maximum (fwhm) ) 6.3 ( 0.2 °C) and
a higher melting temperature (Tm ) 43.5 ( 0.4 °C) compared to
the A:A′ unmodified 15 base-pair DNA duplex (fwhm )10.6 (
0.1 °C, Tm ) 41.3 ( 0.1 °C) (Table 1). Surprisingly, dimers 5 and
6 (Figure 3), where either one or both partners do not have a rigid
core linking the two DNA arms together, also show similarly sharp
melting profiles and higher Tm values (Table 1, entries 11-16) as
in the case of 4. This observation suggests that as long as the linkers
are short enough to keep the DNA arms in close proximity,
cooperative melting can occur readily in the absence of a rigid
linker.
Figure 3. Experimental and theoretical melting curves for A-T6-B:A′-T6-
B′ (0.76 µM) and the experimental melting curve for A:A′ (0.76 µM) in
PBS buffer (10 mM, pH 7.0, 300 mM NaCl). Inset shows the first derivatives
of the melting curves.
model,17 when the salt concentration increases, the Tm difference
between the A:A′ duplex and the dimer 4 also increases (Table 1,
cf. entries 1-3 vs 8-10). Similar comparisons hold for dimers 5
and 6.
We also designed a new rSMDH2 7 (3′-A′-5′-T3CT3-5′-B′-3′) to
hybridize with rSMDH2 3 (3′-A-5′-T3CT3-3′-B-5′) and form a
hairpin-like dimer 8 (3:7). The melting profile of this dimer 8
displays a broader (fwhm ) 9.1 ( 0.3 °C, Figure 4) transition
compared to rSMDH2 dimer 4 (3:3′) (fwhm ) 6.2 ( 0.1 °C). This
again supports our hypothesis that ion-cloud sharing is crucial to
cooperative melting; the parallel orientation of the DNA duplexes
in dimer 4 leads to better overlap between the ion clouds in the
two partner duplexes, and sharper melting profile, in comparison
to that in dimer 8.
From the salt-concentration dependence of the melting temper-
ature of the aggregates (Table 1, entries 5-17), the number of
cooperative duplexes (Nc, defined in eq S2 in the SI) can be
calculated for the various SMDH2 hybridization mixtures (see SI).15
As expected for a cooperative dimer pair, Nc for the caged dimers
averages between 1.6 and 2.1 ( 0.1. Interestingly, while the Tm’s
for the control groups A:A′-T6-B′:B and A:rSMDH2-A′-T3CT3-B′:B
do not differ significantly from that of A:A′, their Nc’s still average
in the range 0.9-1.0 ( 0.1, clearly indicating that there is no
cooperativity between the duplexes.
As a control experiment, the melting profiles of the hybrids
formed between T6-linked A′-T6-B′ and its complementary se-
quences A and B (Table 1, entries 5-7) are broader and have lower
Tm values compared to dimer 6 (Table 1, entries 11-17). As
predicted by the neighboring-duplex/effective-concentration
The formation of DNA dimers such as 4-6 from the dissociated
DNA partners ([a] and [b]) can be viewed as a three-state event
where the first arms come together initially to form [ab1] as a
bimolecular reaction (Figure 5). Hybridization of the second arms
to give [ab2] is nominally unimolecular but can be thought of as a
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C O M M U N I C A T I O N S
strongly favored, is primarily responsible for the sharp melting
behavior in a cooperative, cascade melting mechanism.
In conclusion, we have experimentally demonstrated for the first
time that aggregates with two parallel DNA duplexes in close
proximity can interact cooperatively, consistent with the prediction
by the neighboring-duplex/effective-concentration model.14,15 With
the help of coarse-grain molecular dynamics simulations, we can
now begin to construct accurate analytical models that allow us to
understand the thermodynamic parameters governing the coopera-
tive interactions that occur when DNA duplexes aggregate. This
knowledge will enable researchers to design better DNA-hybrid
materials for a wide range of applications.
Acknowledgment. We thank Drs. Brian Stepp and Erbay Yigit
for their help with the DNA melting and PAGE-gel experiments.
Financial support for this work was provided by the NSF (Grant
EEC-0647560 through the NSEC program) and the NIH (NCI
1U54CA119341-01 through the CCNE program).
Figure 4. Melting curves for dimer 8 (3:7; 0.76 µM) and dimer 4 (3:3′;
0.76 µM) in PBS buffer (10 mM, pH 7.0, 150 mM NaCl). Inset shows the
first derivatives of the melting curves.
Supporting Information Available: Synthetic procedures and
characterization data for rigid small molecule-DNA hybrids; methods
and data for hybridization experiments; theoretical equations for the
analytical model. This material is available free of charge via the
Internet at http://pubs.acs.org.
bimolecular reaction in which the effective local concentration of
the second arms (see SI) is greatly enhanced compared to the
solution concentration. For the reverse (melting) process, the first
step ([ab2] f [ab1]) is therefore entropically disfavored, but the
second step ([ab1] f [a] + [b]) is strongly favored. Additional
factors that favor cooperative melting arise as a result of counterion
clouds which are shared between the two duplexes in [ab2], but
not in later stages, and can be readily modeled by CGMD
simulations.14
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