Flexibility of C3h-Symmetrical Linkers in Tris-oligonucleotide-Based Tetrahedral Scaffolds

Article abstract of DOI:10.1002/cbic.201500436

Flexibility of tris-oligonucleotides is determined by the length of their connecting hydrocarbon chains. Tris-oligonucleotides are branched DNA building blocks with three oligonucleotide arms attached to a C_3h-symmetrical linker core at these chains. Four tris-oligonucleotides hybridise into a tetrahedral nanocage by sequence-determined self-assembly. The influence of methylene, ethylene and propylene chains was studied by synthesising sets of tris-oligonucleotides and analysing the relative stability of the hybridisation products against digestion by mung bean nuclease by using gel electrophoresis. Linkers with ethylene chains showed sufficient flexibility, whereas methylene-chain linkers were too rigid. Tris-oligonucleotides based on the latter still formed tetrahedral scaffolds in intermixing experiments with linkers of higher flexibility. Thus, a new generation of versatile isocyanurate-based linkers was established.

Full text of DOI:10.1002/cbic.201500436

DOI: 10.1002/cbic.201500436
Full Papers
Flexibility of C3h-Symmetrical Linkers in Tris-
oligonucleotide-Based Tetrahedral Scaffolds
Christos Panagiotidis,*[a] Stephanie Kath-Schorr,[b] and Günter von Kiedrowski[a]
Flexibility of tris-oligonucleotides is determined by the length
of their connecting hydrocarbon chains. Tris-oligonucleotides
are branched DNA building blocks with three oligonucleotide
arms attached to a C3h-symmetrical linker core at these chains.
Four tris-oligonucleotides hybridise into a tetrahedral nano-
cage by sequence-determined self-assembly. The influence of
methylene, ethylene and propylene chains was studied by syn-
thesising sets of tris-oligonucleotides and analysing the relative
stability of the hybridisation products against digestion by
mung bean nuclease by using gel electrophoresis. Linkers with
ethylene chains showed sufficient flexibility, whereas methyl-
ene-chain linkers were too rigid. Tris-oligonucleotides based on
the latter still formed tetrahedral scaffolds in intermixing ex-
periments with linkers of higher flexibility. Thus, a new genera-
tion of versatile isocyanurate-based linkers was established.
Introduction
The design strategy in DNA nanotechnology is based on se-
quence-controlled assembly into higher-order hybridisation
motifs. It was influenced by significant contributions from
Seeman[1,2] and Rothemund.[3] DNA nanostructure formation
relies on the specificity of Watson–Crick base pairing as the
major force for structural self-assembly[4] into nano-objects like
hollow polyhedra.[5–7] These constructs are attractive as nano-
scale containers for cargo transport.[8–10] The DNA backbone is
unaltered, thus potentially leading to structural limitations,
such as the fixed persistence length of double strands. Chemi-
cal linkers can change the chemical or structural behaviour of
DNA nanostructures.[11–15] They can, for instance, connect sever-
al linear oligonucleotides together and modify the structure of
a design by acting as flexible hinges.[16–20] The resulting DNA
nanoconstructs can be more compact compared to DNA junc-
tion- and origami-based designs.
cleotides was successfully self-assembled into a dodecahedron
with a diameter of 20 nm.[21]
However, the feasibility of shorter alkyl chains at the linker
core in tris-oligonucleotide-based assembly was not explored.
Here we report a systematic study on the flexibility of methyl-
ene, ethylene, and propylene chains in tris-oligonucleotide-
based tetrahedral scaffolds (Scheme 1B). Four tris-oligonucleo-
tides are sufficient to self-assemble into a DNA tetrahedron
(Scheme 1B), the geometry of which is the simplest case of
a Platonic body. It entails a stiffness unlike the case for the col-
lapsible cube and higher-order geometries, as discussed previ-
ously.[24] In Buckminster Fuller’s principle of tensegrity,[25,26] an
intrinsic tension allows the tetrahedron to maintain structural
stability even if an external force is applied. The highest con-
straint for the alkyl arms at the linker is found in this geometry
and it is therefore the most interesting scaffold to study the
effect of linker flexibility.
We previously established tris-oligonucleotides as branched
DNA building blocks that use C3h-symmetrical covalent linkers
to connect three oligonucleotides with individual sequences.[21]
The achirality of the linker, 1,3,5-trihydroxypropylbenzene,
avoids diastereomeric mixtures in preparation,[22,23] and its pro-
pylene connectors enable unhindered folding of the oligonu-
cleotide arms into polyhedral scaffolds. A set of 20 tris-oligonu-
Results and Discussion
We prepared three phosphoramidites of orthogonally protect-
ed linkers for tris-oligonucleotide synthesis: the “T1 trislinker”
bearing methylene chains, the “T2 trislinker” with ethylene
chains and “T3 trislinker” with propylene chains (Scheme 1A;
see also Scheme S1 and S2 in the Supporting Information). The
synthesis of the T3 trislinker[21] was highly improved by follow-
ing a palladium(II)-catalysed Heck-type coupling reaction of
1,3,5-tribromobenzene with methyl acrylate (following a proce-
dure by Majchrzak et al.)[27] and allyl benzyl ether (Scheme 2).
The new strategy saved up to six synthetic steps compared to
previous efforts.[21,28]
[a] C. Panagiotidis, Dr. G. von Kiedrowski
Lehrstuhl für Organische Chemie I
Bioorganische Chemie, Ruhr-Universität Bochum
Universitätsstrasse 150, 44780 Bochum (Germany)
E-mail: christos.panagiotidis@ruhr-uni-bochum.de
[b] Dr. S. Kath-Schorr
LIMES Institute, Chemical Biology and Medicinal Chemistry Unit
Universität Bonn
All trislinkers were orthogonally protected with 4,4’-dime-
thoxytrityl chloride (DMT-Cl), allyl chloroformate (AOC-Cl) and
(in this work) were phosphitylated in the presence of N,N-diiso-
Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201500436.
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Scheme 2. Synthesis of the T3 trislinker core by a Heck-coupling-type reac-
tion of 1,3,5-tribromobenzene with allylbenzyl ether and one-step hydroge-
nation and hydrogenolysis. a) 3% Pd(OAc)2, 6% Ph3P, TEA, ACN, reflux, 1 d,
37%; b) 40 psi H2,10% Pd/C, MeOH, RT, 16 h, 91%; c) AOC-Cl, Pyabs, THFabs
,
08C–RT, 3 h, 47%; d) DMT-Cl, Pyabs, RT, 16 h, 38%; e) 1.3 equiv 2-Cyanoethyl-
N,N-diisopropylchlorophosphoramidite, 1.3 equiv DIPEA, DCMabs, RT, 2 h.
Scheme 1. A) Linker designs. B) Self-Assembly of tris-oligonucleotides into
a C) tetrahedral scaffold.
propylethylamine (DIPEA) with 2-cyanoethyl-N,N-diisopropyl-
chlorophosphoramidite.[29,30]
The sequences were designed with the DNA sequence gen-
erator program of Feldkamp et al.[31] The output consisted of
a pool of 15-mer sequences with melting points of 58.3–
58.48C at a neutral pH for a 1.25 mm solution in 110 mm
sodium chloride (Table S1). The program used SantaLucia pa-
rameters[32] and the Nearest Neighbour model for duplex
energy calculations. The first and last nucleotides in the se-
quences were G and C, in order to reduce fraying at the blunt
ends. Six unique sequences were picked and assigned together
with the six complementary sequences onto a Schlegel repre-
sentation of a tris-oligonucleotide-based tetrahedral scaffold
(Figure 1).
Figure 1. Schlegel representation of a tris-oligonucleotide-based tetrahedron
and assigned sequences. Each tris-oligonucleotide is numbered at the tris-
linker, with the 3’-ends at the linker. Sequence design and numbering are
identical for all sets.
with the starter nucleoside attached by a succinic ester link-
age.[34] The first strand was synthesised in the 5’!3’ direction
by using reverse amidites, and followed by coupling of one
trislinker per set (Figure S1). Subsequently, the second arm was
synthesised after linker detritylation, and then the third arm
was synthesised after palladium-catalysed removal of the AOC
protecting group (synthesis direction changed to 3’!5’ for the
second and third arm).
Three sets of four tris-oligonucleotides were prepared on a
DNA synthesiser on a 1.3 mmol scale by using a previously es-
tablished protocol[21] with commercial polystyrene supports,
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Figure 2. Gels for fully assembled A) T1, B) T2 and C) T3 showing all dimer and trimer permutations (e.g., “12”=monomers 1 and 2; “123”=monomers 1, 2
and 3). Single monomers “1” and “4” added for reference; the last band is the final tetramer; 1.25 mm in hybridisation buffer; native 3% Agarose 1000, 1”
TBE, 100 V, 75 min; GelStar nucleic acid gel stain (in-gel).
Following automated synthesis and cleavage from the solid
support, each tris-oligonucleotide was purified by preparative
denaturing PAGE. The products were manually extracted, de-
salted by size-exclusion chromatography with NAP-25 columns
and quantified by UV spectroscopy (yield: 26 to 208 nmol (2–
16%) after purification). Aqueous stock solutions were pre-
pared at 125 mm, and the masses were confirmed by MALDI-
TOF-MS (Table S2). The purity of each product was confirmed
by agarose gel electrophoresis (Figure S3).
The stock solutions were diluted to 1.25 mm for assembly ex-
periments with a hybridisation buffer (10 mm HEPES, pH 7.5,
with 100 mm NaCl). Tris-oligonucleotides were combined in
equimolar amounts for each set by denaturing (5 min at 958C),
annealing (0.38CsÀ1; 5 min at 408C; ~208C below the theoreti-
cal melting point of single strands) and a final low-temperature
step (0.18CsÀ1; 15 min at 08C; Table S3).
Self-assembly (Figure 2) was carried out for every possible
Figure 3. Digestion of trimer 123 of the T3 set and tetramers T1, T2, T3;
“+E”: incubation with 5 U mung bean nuclease for 10 min at 308C;
1.25 mm; native 3% Agarose 1000, 1” TBE, 100 V, 2 h; GelStar nucleic acid
combination of building blocks. The assembly was analysed by
native agarose gel electrophoresis for each set to test for
errors in the sequences leading to the assembly of all four tris-
oligonucleotides. Six distinct binary combinations lead to
dimers connected at one arm (Figure 2, “12” to “34”; numbers
according to Figure 1), four trimer combinations containing
three double stranded arms (“123” to “234”) and one unique
tetramer combination with six double strands. Two of the four
monomers (“1” and “4”) were added as references. The bands
showed full addressability of each arm and no sequence errors.
The faint higher-order bands and smearing can be explained
by mismatching side reactions and the formation of higher ag-
gregates (most noticeable in the T2 set). A noticeable band
(~180 bp) above the tetramers was assigned to a cube-shaped
side product (Scheme S3). All mixtures assembled into tetram-
ers (“T1”, “T2”, “T3”) with the expected electrophoretic mobility
below the 100 bp marker.
gel stain (in-gel).
present in the test trimer subset “123”. The T3 tetramer band
(Figure 3) showed no digestion pattern, as previously observed
for fully paired tris-oligonucleotide nanostructures such as do-
decahedra. Thus, T3 contained only non-digestible double
strands needed for a fully assembled 3D object. T1, in contrast,
produced single strands upon digestion. It can be concluded
that the T1 trislinker alone is too rigid to enable strain-free
folding into a tetrahedral scaffold.
It was, however, possible to successfully intermix T1 mono-
mer tris-oligonucleotides with the more flexible T3 monomers
to result in more-stable constructs (Figure 4). The setup started
with the assembly of the four T1 monomers (“40”: four units of
set T1 and zero units of T3) and replacement stepwise with
the analogous tris-oligonucleotide of the T3 set. Stability was
again analysed with mung bean nuclease. The substitution of
just one tris-oligonucleotide (Figure 4, “31+E”) resulted in
a major increase in stability (minimal digestion). Apparently,
Treatment with mung bean nuclease (Figure 3, “+E”)
showed distinct digestion patterns for trimer “123” (of the T3
set) and the T1 tetramer, whereas no change in electrophoretic
mobility was observed for the T2 and T3 tetramers. Mung
bean nuclease preferably digests single-strands, which were
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thus be prepared faster and at lower cost, thus alleviating a
bottle-neck in the bulk preparation of tris-oligonucleotides.
Flexibility and stability of the TN trislinker in tris-oligonucleo-
tides was tested as for the T1–T3 sets. A set of four tris-oligo-
nucleotides was synthesised and studied in self-assembly
experiments (Figure S4). The assembled product of all four tris-
oligonucleotides (Figure 5, “TN”) showed no digestion for at
Figure 4. Mixed assemblies between the T1 and the T3 set; 1.25 mm; “+E”:
5 U mung bean nuclease for 10 min at 308C; native 3% Agarose 1000, 1”
TBE, 100 V, 60 min; GelStar nucleic acid gel stain (in-gel).
Figure 5. Digestion of trimer 123 of the TN set and tetramer TN; “+E”: 5 U
mung bean nuclease for 5 or 20 min at 308C; 1.25 mm; native 3% Agarose
1000, 1” TBE, 100 V, 65 min; GelStar nucleic acid gel stain (in-gel).
the more flexible T3 propylene chains were able to decrease
the strain caused by the rigidity of the T1 methylene chains
and allowed the formation of a stable tetrahedron. Stability
further increased for a 50:50 mix (Figure 4, “22+E”). The tetra-
mer of the T2 set (Figure 3) behaved similarly to the T3 set
with respect to enzymatic digestion. Thus, a fully folded tetra-
hedral scaffold is assumed.
least 20 min (Figure 5, “TN+E”), unlike the test trimer
(Figure 5, “123+E”). All single strands were therefore hybri-
dised, thus also indicating a fully closed tetrahedral scaffold
and confirming the findings for the T2 set. The TN set was fully
intermixable with the T3 set and had a similar stabilising effect
in T1 intermixes (Figure S5). Isocyanurate-based tris-oligonucle-
otides and T1 decompose much faster than T2 and T3, when
stored in aqueous solution at >08C, but are sufficiently stable
for at least one year when stored at À208C.
Because the ethylene chains proved sufficiently flexible,
a new generation of trislinkers (“TN”) was established by using
commercial
1,3,5-tris(2-hydroxyethyl)-N,N’,N’’-isocyanurate
(THEIC), which has a geometry similar to that of the T2 tris-
linker core. Synthesis of the linker core is omitted, and the TN
trislinker thus only requires the introduction of the protection
groups and the phosphoramidite (Scheme 3). The linker can
Scheme 3. Complete synthesis of the TN trislinker including the orthogonal protection group chemistry and the phosphoramidite reaction. a) 0.8 equiv DMT-
Cl, 1:1 DMFabs/Pyabs, RT–608C, 16 h, 30%; b) 0.8 equiv AOC-Cl, 1.2 equiv Py, THFabs, 08C–RT, 4 h, 30%; c) 2 equiv 2-Cyanoethyl-N,N-diisopropylchlorophosphor-
amidite, 2 equiv DIPEA, DCMabs, RT, 2 h, 45%.
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was then heated in a microwave to boiling, and then heated in
short intervals (~10 s) until the agarose was fully dissolved. GelStar
Nucleic Acid Stain (1 mL; Lonza) was added for in-gel staining, and
the mixture was stirred until it cooled to 40–508C. The warm solu-
tion was then cast in a 7”7 cm tray placed in a gel caster (Sub-
Cell GT UV-Transparent Mini-Gel Trays, Bio-Rad) and either an 8- or
a 15-well comb was then added into the gel. The gel was covered
in aluminium foil and allowed to cool to room temperature. The
gels had a thickness of 4–7 mm. The gels had a pre-run for 15 min
at 100 V and at ~88C in a cooling chamber. Gel loading buffer
(1 mL; Sigma–Aldrich) was added to each sample. GeneRuler Low
Range DNA Ladder (25–700 bp; 50 mg; Life Technologies) served as
an internal reference for DNA and as an external reference with
the added dyes. Electrophoresis was executed at 100 V and ~88C
in a cooling chamber for 30–120 min.
Conclusion
We have studied the effect of short alkylene chains of linkers
in tris-oligonucleotide-based tetrahedral scaffolds by hybridisa-
tion and digestion experiments. Unlike methylene, ethylene
chains at the linker are sufficiently flexible to allow folding into
tetrahedral nano-objects. THEIC provides a new generation of
cheap and versatile C3h-symmetrial trislinkers, compatible and
sufficiently flexible for use in tris-oligonucleotide-based nano-
structures.
Experimental Section
General: See the Supporting Information for organic preparations
and experimental procedures. All solvents and chemicals were of
analytical grade unless otherwise noted. NMR spectra were record-
ed with DPX-200, DPX-250 and DRX-400 spectrometers (Bruker).
MS analysis was performed on an Autoflex spectrometer (Bruker)
equipped with an LTB MNL106 nitrogen-laser. Tris-oligonucleotides
were synthesised on a Gene Assembler Plus machine (Pharmacia
Biotech). The custom 3’ solid support was based on Primer Support
200 Amino (GE Healthcare) with 3’-end dC- and dG-starter nucleo-
sides (~50 mmolgÀ1) on a 1.3 mmol scale (previously synthesised ac-
cording to ref. [34]). Synthesis, purification and characterisation of
tris-oligonucleotides were according to ref. [21]. Mung bean endo-
nuclease (40 unitsmLÀ1) was purchased from Roboklon (Berlin, Ger-
many).
Enzymatic digestion: DNA (4 mL, picomol) was diluted in ddH2O
(5 mL) with reaction buffer (1 mL, 30 mm sodium acetate, pH 5.0,
50 mm NaCl, 1 mm ZnCl2, Roboklon) and 0.125 mL mung bean nu-
clease (In storage buffer containing 10 mm Tris·HCl, pH 7.5, at 22
8C, 0.1 mm zinc acetate, 50% (v/v) glycerol, Roboklon) was added.
Samples were incubated at 308C on a thermocycler for between
5 and 30 min. The samples were transferred to a cooling chamber
(~88C), mixed with gel loading buffer (1 mL; Sigma–Aldrich) and
placed as soon as possible onto the gel.
Acknowledgements
We thank Michael Wüstefeld for automated oligonucleotide syn-
theses and protocol development for tris-oligonucleotides. We
thank Jan Castonguay for preliminary work on the synthesis of
a tris-oligonucleotide based on the T2 linker as it was previously
described in his bachelor thesis.
MALDI mass analysis: A sample (1.5 mL, 1–10 pmolmLÀ1) was
mixed with a few beads of an ion-exchange resin (NH4+) on top of
a plastic paraffin film (Parafilm; Pechiney Plastic Packaging). After
~15 s, the supernatant (1 mL) was mixed with 3-HPA (3 mL, Fluka)
on top of the parafilm (matrix: 3-HPA (80 mg) was diluted in
ddH2O/ACN (1:1; 2 mL) and stored at À208C). An aliquot of the
mixture (2–4 mL) was placed on the steel target (MTP 384, Bruker
Daltonics) and allowed to dry at room temperature for at least
30 min.
Keywords: DNA structures · isocyanurate · nanotechnology ·
self-assembly · tris-oligonucleotide
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Manuscript received: August 27, 2015
Accepted article published: November 23, 2015
Final article published: December 17, 2015
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