DNA minicircles with gaps for versatile functionalization

Article abstract of DOI:10.1002/anie.200704004

(Figure Presented) Doing the rounds: A highly straightforward approach has been developed that provides access to DNA minicircles containing a 21-mer single-stranded gap region of defined sequence (see picture). The single-stranded domain within a small c

Full text of DOI:10.1002/anie.200704004

Angewandte
Chemie
DOI: 10.1002/anie.200704004
DNA Structures (1)
DNA Minicircles with Gaps for Versatile Functionalization**
Goran Rasched, Damian Ackermann, Thorsten L. Schmidt, Peter Broekmann,
Alexander Heckel, and Michael Famulok*
The programmable self-association of molecular units into
higher ordered structures plays a key role in the bottom-up
construction of nanomaterials.[1] Crucial for the successful
supramolecular assembly of nanoobjects, however, is the
choice of the functional molecular units themselves. Nucleic
acids have emerged as a convenient target for these purposes
because of their unique properties in molecular recognition
and the ease by which oligonucleotides can be accessed
synthetically.[2,3] The assembly of DNA nanoobjects with
defined two- or three-dimensional geometries requires either
rigid subunits, such as in double crossover (DX) or paranemic
crossover (PX) elements,[3] or inherently rigid triangular
shapes such as tetrahedra or bipyramids.[4]
From a structural point of view, DNA minicircles are
probably the simplest rigid objects with a nanometer size.
Small DNA circles were first prepared by designing two 21-
mer DNA precursor sequences which, upon hybridization and
ligation, resulted in a statistical distribution of DNA mini-
circles containing 105, 126, 147, and 168 base pairs (bp).[5a]
Atomic force microscopy (AFM) analysis of 168-bp mini-
circles confirmed their smooth circular structure without any
ring deformation or supercoiling.[6,7] These features predes-
tine them as building blocks for the assembly of objects on the
nanometer scale. However, a major drawback of DNA
minicircles in the construction of higher ordered DNA
architectures is their unbranched, continuously double-
stranded (ds) nature that prevents the guided aggregation of
multiple rings. Furthermore, the statistical assembly of the
short oligonucleotides that were applied in the known
strategies for the synthesis of DNA minicircles[5–7] prevents
the controlled introduction of “customized” sequences into
the circle that can serve as defined handles for the self-
assembly of multiple rings.
We recently reported the assembly of two DNA mini-
circles, guided by a “strut” of Dervan-type polyamides that
specifically held two rings together by binding to different 9-
mer double-stranded “custom” sequences that were present
in each 168-mer ring exactly once.[8] Here we report on the
construction of minicircles that contain a customized single-
stranded gap sequence at a defined position. The gap can
serve as a versatile site for the modification of DNA
minicircles, thereby allowing, for example, their guided
modification with functional groups through hybridization
with synthetic oligonucleotides.
The key step in the preparation of DNA minicircles with
sequence-specific functionalization focuses on a preformed
incomplete minicircle MCgap containing a 21-nucleotide
single-stranded (ss) gap region (Figure 1). Hybridizing and
Figure 1. Design of gap-containing DNA minicircles. A) MCgap (left):
DNA minicircle with a single-stranded gap region for sequence-specific
hybridization with a complementary functionalized (F) oligonucleotide
(ONfunct). MCfunct (middle): functionalized DNA minicircle afforded by
either hybridization or direct ligation with an ONfunct. MCfull (right):
completely double-stranded DNA minicircle with a 21-bp custom
sequence (red). B) Forward (f) and reverse (r) sequences of the
dsDNA segments (a, b, g) with individual 5’ overhangs (color coded)
ensuring the assembly of 168-bp rings or multiples thereof. The red-
colored 21-bp region in segment g contains the customized sequence.
Modified bases in ONfunct are framed in black.
[*] G. Rasched, Dr. D. Ackermann, T. L. Schmidt, Dr. A. Heckel,
Prof. Dr. M. Famulok
Universität Bonn, LIMES-Life and Medical Science
Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany)
Fax: (+49)228-73-4809
E-mail: m.famulok@uni-bonn.de
Dr. P. Broekmann
Universität Bonn
Institut für Physikalische und Theoretische Chemie
Wegelerstrasse 12, 53115 Bonn (Germany)
[**] This work was funded by grants to M.F. from the Sonderfor-
schungsbereich 624 (From the Design of Chemical Templates
Towards Reaction Control). We gratefully acknowledge help from H.
Rasched in generating the computer model of the minicircles.
ligating this gap-containing minicircle with any complemen-
tary 21-mer oligonucleotide ONfunct can lead to a variety of
differently functionalized DNA minicircles MCfunct in a highly
straightforward manner.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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The prerequisite in the design of DNA minicircles is the
closed DNA minicircles, lane 3 was excised completely and
subjected to PAGE in the second dimension in the presence of
chloroquine diphosphate.[10] As shown in Figure 2B, only
band 4 splits up. Figure 2C supports the open-chain nature of
the faster migrating spot, which is sensitive to digestion with
nuclease Bal31, whereas the slower one represents the desired
168-bp DNA minicircle. This result shows that 168-bp DNA
minicircles tolerate a 21-bp segment that lacks any A-tract
arrangement. Interestingly, the formation of the gap-contain-
ing DNA minicircle MCgap was also possible under these
conditions, although we observed a slight increase in the
amount of side products compared to the formation of MCfull
(data not shown), presumably because the short precursor
sequences gf20 and gf15 only form weak duplexes. All the
ligation experiments thus yielded a distinct band, either
representing MCfull or the so far unknown gap-containing
repetition of A-tracts in the forward strands (af, bf, and gf;
Figure 1) since they cause a bending of the helical axis.[9] To
obtain circularly bent DNA double strands, cooperative
bending of all A-tracts is required. This only occurs if d(A)5
and d(A)6 A-tracts are arranged in tandem repeats alternating
with d(N)5 random base pairs.[5] However, for our purposes,
the synthesis of minicircles starting from two 21-mer pre-
cursor DNA strands is not suitable since the incorporation of
a gap region is not possible.
To achieve this, we constructed 168-bp DNA circles from
three segments a, b, and g (Figure 1). All segments consist of
a DNA double strand of 51 bp having five base overhangs on
their 5’ ends (Figure 1B). The choice of the sticky ends is the
crucial specifying factor to selectively form the 168-bp DNA
circles and no other sized rings. Thus, the segments are
designed so that they hybridize only in the defined cyclic
order (a!b!g!a). In addition, the 21-mer gap no longer
follows the A-tract arrangement, since the incorporation of
any custom sequence is desired.
minicircle MCgap
.
To further confirm the circular nature of the minicircles,
MCfull and MCgap were visualized by AFM. Figure 3A shows
After performing the ligation steps (Supporting
Information, Figure S2), the DNA minicircles were analyzed
and purified by native two-dimensional polyacrylamide gel
electrophoresis (2D PAGE).[10] In the first dimension, the
products were separated according to their size (Figure 2A).
Lane 3 shows the crude ligation product of the DNA
minicircle MCfull. Besides the product (band 4), the mono-
mers (band 1) and dimers (band 2) are still present in the
mixture, probably because of incomplete phosphorylation. To
separate c-shaped (open-ring) DNA side products from
Figure 2. Identification of 168-mer DNA minicircles by 2D PAGE.
A) The one-dimensional gel analysis separates the ligation products on
the basis of the length of their base-pair segments. Lane 1: segment
a; lane 2: products of the ligation of segments a+b; lane 3: products
of the ligation of a, b, and g. Band 1: one segment, monomer product;
band 2: ligation product of segment dimers; band 3: unknown side
product or secondary structure; band 4: trimer ligation products
(168 bp). B) Two-dimensional gel analysis of lane 3 separates the
circular (O-form) from the bent open (C-form) DNA. C) Two samples
of the band 4 product (lane 3, gel (A) separated in a second dimension
in the presence (left) and absence (right) of nuclease Bal31.
Figure 3. In situ atomic force microscopy (AFM) scans of DNA mini-
circles. A) MAC mode[11] AFM scan of DNA minicircles MCfull adsorbed
on mica surfaces in the presence of 10 mm NiCl2. The fully double-
stranded DNA minicircles containing the custom sequence show no
distortion and are of uniform shape and size. B) The same experiment
with gap-containing DNA minicircles (MCgap). Zoomed sections show
DNA minicircles with a narrow region (yellow arrows). The model of a
MCgap (top right) shows the dimensions of the single-stranded region
in relation to the ring. Green arrows: linear bent DNA fragments.
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Angewandte
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the uniformly circular structure of the fully double-stranded
MCfull. Although these rings bear a 21-mer custom sequence
lacking poly-A tracts, they do not show any ring deformation.
The diameter of the rings is roughly 20 nm, which matches
that of 18.2 nm expected for DNA minicircles with 168 bp
(Supporting Information).
double helix without affecting the DNA structure.[13] Fur-
thermore, a slight stabilization of the DNA douplex is
observed when the aminopropargyl residues of the spacer
moiety are attached to pyrimidine nucleobases.[14] These
considerations, together with the fact that anthracene resi-
dues can potentially intercalate into dsDNA, led to the use of
ONfunct (Figure 1B) as the pilot oligonucleotide for the
functionalization of DNA minicircles.
For the synthesis of phosphoramidite 1 (Figure 4A), we
first derivatized the C5-position by a Sonogashira coupling
reaction between 5-iododeoxyuridine and N-prop-2-ynyltri-
fluoroacetamide (Supporting Information, Scheme 1).[13]
After changing the protecting groups, anthracene-9-carba-
mido-e-aminocapronic acid was attached to the amino group
using N,N,N’,N’-tetramethyl-O-(N-succinimidyl)uronium tet-
rafluoroborate (TSTU) as a coupling reagent.[15] The obtained
functionalized nucleoside was converted into the correspond-
ing phosphoramidite 1 under standard conditions.
ONfunct was prepared by automated solid-phase DNA
synthesis. Phosphoramidite 1 was successfully incorporated
into the 21-mer oligonucleotide at positions 6 and 16, with
coupling efficiencies between 96–98%, as determined by
analysis of the amount of trityl. After cleavage of the product
from the solid support and removal of all base-labile
protecting groups, ONfunct was purified by reverse-phase
HPLC. ESI-MS studies on ONfunct confirmed the successful
incorporation of two anthracene-modified deoxyuridine units.
We then hybridized ONfunct with the DNA minicircle
MCgap, thereby resulting in the highly sequence specific
functionalized DNA minicircle MCfunct (Figure 4B). The
relative gel mobilities of the three purified DNA minicircles
MCfull, MCgap, and MCfunct were examined (Figure 5). All three
In contrast, the topology of the gap-containing minicircles
MCgap markedly differs from that of MCfull (Figure 3B).
Clearly, these rings exhibit a higher degree of distortion,
presumably because of an increased flexibility of the 21-mer
single-stranded region, compared to the MCfull rings. Inter-
estingly, all the circles show a single constriction along the
outline of the ring, likely resulting from the single-stranded
region, a phenomenon that is never observed for MCfull. While
this topology dominates in the MCgap scan, some linear bent
shapes are also present, either because of damage induced by
mechanical stress by the cantilever tip or exposure to shear
forces during sample preparation and purification. Similar
effects have been described previously in dsDNA scans.[7,11,12]
Having confirmed the circular structure of MCgap by
AFM, we next sought to equip MCgap with additional
chemical functionality by hybridizing it to a chemically
functionalized DNA fragment. As an initial proof-of-princi-
ple, we designed a 21-mer oligonucleotide that was comple-
mentary to the gap sequence and contained two anthracene
moieties at defined positions, attached to the C5-position of
deoxyuridine residues through a spacer (Figure 4). The
anthracene and the nucleobase were chosen for synthetic
convenience and because these substituents point outside the
Figure 5. Analysis of the different DNA minicircles MCgap, MCfunct, and
MCfull by native PAGE.
circles showed the characteristic slow migration behavior
known for DNA minicircles.[5] As expected, MCgap has a
higher mobility than the fully hybridized MCfull and MCfunct
.
Although these latter two species have the same charge and
the same nucleobase sequence, they separate on the gel as a
result of the presence of the two anthracene residues in
MCfunct
.
In summary, we have established a straightforward
method for preparing highly sequence specific functionalized
DNA minicircles based on chemical synthesis, the program-
med self-assembly of DNA oligonucleotides, and enzymatic
ligation. Crucial for that is the design of the precursor
sequences in the cyclic order (a!b!g!a), which not only
Figure 4. Anthracene-functionalized monomer and ONfunct. A) Phos-
phoramidite (1); DMT=4,4’-dimethoxytriphenylmethyl. B) The func-
tionalized oligonucleotide ONfunct hybridizes with its complementary
region in the gap-containing minicircle MCgap
.
Angew. Chem. Int. Ed. 2008, 47, 967 –970
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969

Communications
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Received: August 30, 2007
Published online: December 3, 2007
Keywords: DNA · DNA structures · functionalized DNA ·
.
nanostructures · scanning probe microscopy
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