A supramolecular DNA self-assembly based on β-cyclodextrin-adamantane complexation as a bioorthogonal sticky end motif

Article abstract of DOI:10.1039/c3cc43109f

We propose linear end-to-end assemblies of short DNA duplexes based on β-cyclodextrin-adamantane complexation. The assembled duplexes exhibited increased T_m values compared with those of the corresponding natural hybrids. Competition experiments with external guest molecules showed a substantial decrease in T_m of the terminal modified duplexes, suggesting the viability of inter-duplex complexation.

Full text of DOI:10.1039/c3cc43109f

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A supramolecular DNA self-assembly based on
b-cyclodextrin–adamantane complexation as a
bioorthogonal sticky end motif†
Junya Chiba,*a Ayumi Sakai,a Syogo Yamada,a Kazuhisa Fujimotob and
Masahiko Inouye*a
Cite this: DOI: 10.1039/c3cc43109f
Received 26th April 2013,
Accepted 29th May 2013
DOI: 10.1039/c3cc43109f
www.rsc.org/chemcomm
We propose linear end-to-end assemblies of short DNA duplexes DNA dumbbell was recently reported, in which linear end-
based on b-cyclodextrin–adamantane complexation. The assembled to-end assemblies of 10–30 dumbbell monomers as well as
duplexes exhibited increased Tm values compared with those of bundles of the linear polymers formed.6 However, well-defined
the corresponding natural hybrids. Competition experiments with H–G complexation has not been employed for the construction
external guest molecules showed a substantial decrease in Tm of of linear DNA supramolecular polymers. If such H–G complexa-
the terminal modified duplexes, suggesting the viability of inter- tion could be utilized as a sticky end in DNA termini, this
duplex complexation.
bioorthogonal technique may offer an additional strategy for
constructing DNA nanoarchitecture. Because we can choose a
DNA is a unique nanoconstruction tool for rational molecular H–G pair from various types of H–G combinations, we may
architectures because of its ease of synthesis and base-pairing control the association and the external-stimulus dissociation
loyalty with sequence programmability. Attachment of artificial of supramolecular DNA polymers. Here we report an end-to-end
functional components to natural DNA has led to evolution of type supramolecular DNA self-assembly based on b-cyclodextrin–
fields of supramolecular chemistry, DNA nanotechnology, and adamantane (b-CD–Ad) association attached to both the ends of
chemical biology.1 An attractive approach to set up function- DNA duplexes.
alized DNA is to conjugate natural DNA scaffolds with host–
In our previous sensors, CDs as the guest-binding moiety
guest (H–G) chemistry. We have reported DNA duplex-based, were attached to the termini of DNA duplexes, and the bis-Ads
versatile fluorescent sensors consisting of three functional guest molecule was externally added to the DNA solution. We
regions, i.e., host molecules such as crown ethers and cyclo- expected that direct introduction of CD and Ad groups into
dextrins (CDs), DNA, and fluorescent pyrenes.2 Presence of the both the 50-ends of ds-DNA would induce end-to-end assembly
corresponding guest molecules induced emission switching of the short ds-DNA through inter-duplex b-CD–Ad complexa-
from a monomer to an excimer of the pyrenes due to the tion, affording a linear supramolecular structure (Fig. 1 and
DNA hybridization promoted by H–G association.
Fig. S1, ESI†). Thus, we designed 50-b-CD- and 50-Ad-modified
Among the various DNA structures, one-dimensional long complementary oligodeoxynucleotides (ODNs). According to our
structures assembling with short oligomers are fundamental previous procedure,2 the ODNs were synthesized by Huisgen
structural motifs of DNA nanoarchitecture. Traditional approaches reaction7 between the alkynylnucleoside residue8 reported by
rely on Watson–Crick base pairing using thermodynamic assembly
of ‘‘sticky ends’’.3 Kinetically controlled self-assembly of
natural oligomers was also reported to build one-dimensional
linear DNA.4 End-to-end double helix assembly based on hydro-
phobic interaction of terminal base pairs was observed at high
concentrations in single crystals and in liquid crystals.5 In dilute
solution, hydrophobic self-assembly of a perylenediimide-linked
a Graduate School of Pharmaceutical Sciences, University of Toyama,
Sugitani 2630, Toyama 930-0194, Japan. E-mail: chiba@pha.u-toyama.ac.jp,
inouye@pha.u-toyama.ac.jp
b Department of Applied Chemistry and Biochemistry, Kyushu Sangyo University,
Fig. 1 (a) Schematic illustration of an end-to-end self-association of DNA hybrids
based on b-cyclodextrin–adamantane (b-CD–Ad) complexation. (b) Sequences of
ODNs 1, 10, 1CD, 10Ad, 2CD, and 20. X represents nucleoside residues of Huisgen
products between alkynylnucleoside and azido derivatives of b-CD 3 or Ad 7.
Matsukadai 2-3-1, Higashi-ku, Fukuoka 813-8503, Japan
† Electronic supplementary information (ESI) available: Detailed synthetic pro-
cedures, Fig. S1–S3, and Table S1. See DOI: 10.1039/c3cc43109f
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point at 240 nm, which indicated that only two absorptive
species, the monomeric and the associated states of 10, exist
in the solution. In the curve-fitting analysis, a well-fitted
theoretical curve could be drawn on the basis of the assump-
tion of 1 : 1 complexation between 10 and 11, and the dissocia-
tion constant was obtained to be Kd = 1.5 ꢁ 10ꢀ7 M (Fig. S3b,
ESI†). In addition, isothermal titration calorimetry (ITC) measure-
ments were carried out for the same combination of 10 and 11,
which afforded a Kd of 8.7 ꢁ 10ꢀ7 M (DG293 = ꢀ8.2 kcal molꢀ1
,
DH = ꢀ9.7 kcal molꢀ1, and DS = ꢀ5.1 cal Kꢀ1 molꢀ1) (Fig. S3c,
ESI†). These two dissociation constants are comparable to the
reported Kd values for complexes between b-CD and phenyl-
substituted Ad derivatives in water.11 Considering from the
B10ꢀ7 M order of the Kd values, we concluded that almost all of
the b-CD and Ad moieties would form the inter-strand complex
between 1CD and 10Ad in the UV-melting experiments in the
concentration range of 10ꢀ5 M.
Scheme 1 (a) HOCH2CH2OH, EDC, DMAP, CH2Cl2; 93%, (b) p-TsCl, DMAP,
pyridine; 90%, (c) NaN3, DMF; 98%, (d) HO(CH2CH2O)8CH3, EDC, DMAP, CH2Cl2;
70%, (e) CCl3CO2H, CH2Cl2; 88%, (f) 3, CuCl, H2O; 89%.
us and azide derivatives of b-CD 3 (ref. 9) or Ad 7. The crude
ODNs were purified by reverse-phase HPLC and characterized
by matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass measurements (see ESI†). For an azide
derivative of Ad, 7 was newly synthesized in good overall yield
from 4-adamantyl benzoic acid 4 (ref. 10) via esterification,
tosylation, and azidation steps (Scheme 1).
Next, we attempted competition experiments for the
duplex formation on 1CD–10Ad, 1–10Ad, 1CD–10, and 1–10 with
1-adamantanecarboxylic acid or b-CD as a competitor (Table 1).
Duplexes lacking in CD (1–10Ad) or Ad (1CD–10) as well as
natural 1–10 showed no obvious Tm changes upon addition
of the competitors (entries 4–12). On the other hand, sub-
stantial Tm decreases were observed for 1CD–10Ad by the
addition of 1 mM 1-adamantanecarboxylic acid or b-CD
(entries 2 and 3), verifying the inter-duplex b-CD–Ad complexa-
tion for 1CD–10Ad.
First, we evaluated thermal stability of the duplexes for
1CD–10Ad and the corresponding natural 1–10. Based on the
first derivative profiles of the UV-melting curves of the two
duplexes (Fig. S2, ESI†), melting temperatures (Tm) of 45 1C for
1CD–10Ad and 27 1C for 1–10 were obtained (see also Table 1,
entries 1 and 10). The drastic increase in the thermal stability
(DTm = 18 1C) suggested that b-CD and Ad moieties in 1CD–10Ad
significantly take part, probably form H–G complexes, in the
higher-order structure formation between 1CD and 10Ad.
In order to confirm the definite complexation between the
b-CD and Ad moieties in the measurement concentration range
of 10ꢀ5 in water, we prepared two model compounds 10 and 11
(Scheme 1). The self-association of 10 was judged to be negligible
at r60 mM by a UV dilution experiment, so that the following
titration assays were carried out below that concentration. The
addition of 11 into an aqueous solution of 10 (40 mM) caused
significant spectral change in UV measurements; the absorbance
at 245 nm gradually decreased, while that at 210 nm increased
(Fig. S3a, ESI†). This alteration was accompanied by an isosbestic
We prepared two natural ODNs of 30-CTGACTGTTTGGGG-50
ꢀꢀꢀꢀꢀꢀꢀ
and 50-AAACCCCGACTGAC-30 as the corresponding natural
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
ꢀ ꢀ
sticky-end polymerizing component with a 7 mer core duplex.
In the ODNs, italicized and underlined residues are comple-
mentary respectively, and the underlined combination has the
same sequence as the core 7 mer of 1–10. A thermal denatura-
tion experiment for the duplex of 30-CTGACTGTTTGGGG-50/
ꢀꢀꢀꢀꢀꢀꢀ
50-AAACCCCGACTGAC-30 ([ssDNA] = 8 mM, 20 mM Tris-HCl
ꢀꢀꢀꢀꢀꢀꢀ
(pH 8.0), 100 mM NaCl, 1 1C minꢀ1, path length = 10 mm)
showed the increased thermal stability (DTm = 5.5 1C) compared
with that of the core 7 mer 1–10. The adjacent core 7 mer-
duplexes in the natural sticky-end polymer would interact with
each other by end-to-end stacking of the terminal base pairs.
This may be one of the reasons for the observed stabilization as
seen in the increased thermal stability of dangling ends. At the
present stage, it is difficult to clearly envisage the molecular
basis for the reason why 1CD–10Ad showed further thermal
stability beyond the corresponding natural sticky-end polymer.
We speculate that the CD–Ad complexes interact with the
terminal base pairs of the core duplexes in good orienta-
tion avoiding destabilization based on steric hindrance of the
complexes.
Table 1 Competition experiments on AdCO2H or b-CD for duplexes 1CD–10Ad,
1–10Ad, 1CD–10, and 1–10
Entry
Duplex
Additive
Tm (1C)
1
2
3
4
5
6
7
8
1CD–10Ad
1CD–10Ad
1CD–10Ad
1–10Ad
1–10Ad
1–10Ad
1CD–10
1CD–10
1CD–10
1–10
—
45
38
33.5
32
32
30.5
30.5
31
30
27
1 mM AdCO2H
1 mM b-CD
—
1 mM AdCO2H
1 mM b-CD
—
1 mM AdCO2H
1 mM b-CD
—
1 mM AdCO2H
1 mM b-CD
To investigate the effect of the lengths of DNA helices, we
further prepared several b-CD- and Ad-modified ODNs (12CD,
120Ad, 13CD, 130Ad, 14CD, 140Ad, 15CD, 150Ad, 16CD, and
160Ad) with different 5–14 mer lengths (see ESI† and Table S1).
All of the modified duplexes 12CD–120Ad (14 mer), 13CD–130Ad
(10 mer), 14CD–140Ad (8 mer), 1CD–10Ad (7 mer), 15CD–150Ad
(6 mer), and 16CD–160Ad (5 mer) exhibited increased Tm values
compared with those of the corresponding natural hybrids 12–120,
9
10
11
12
1–10
27
26.5
1–10
[ssDNA] = 10 mM, 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 1C minꢀ1
,
path length = 10 mm.
c
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structure and additional advancements of this bioorthogonal
technique are now in progress for constructing DNA nano-
architecture in which an additional strategy by H–G chemistry
is utilized.
The authors acknowledge Prof. N. Nakashima and Dr T. Fujigaya
for the ITC measurements at Kyushu University.
Notes and references
Fig. 2 Duplex lengths versus Tm values for natural (blue) and modified (gray) DNA.
The red bars indicate the DTm values. [ssDNA] = 10 mM, 20 mM Tris-HCl (pH 8.0),
100 mM NaCl, 1 1C minꢀ1, path length = 10 mm.
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