“Printing” DNA Strand Patterns on Small Molecules with Control of Valency, Directionality, and Sequence

Article abstract of DOI:10.1002/anie.201809251

The incorporation of synthetic molecules as corner units in DNA structures has been of interest over the last two decades. In this work, we present a facile method for generating branched small molecule-DNA hybrids with controllable valency, different seq

Full text of DOI:10.1002/anie.201809251

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: “Printing” DNA Strand Patterns on Small Molecules with Control
of Valency, Directionality and Sequence
Authors: Tuan Trinh, Daniel Saliba, Chenyi Liao, Donatien de
Rochambeau, Alexander Lee Prinzen, Jianing Li, and Hanadi
Farouk Sleiman
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201809251
Angew. Chem. 10.1002/ange.201809251
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10.1002/anie.201809251
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“Printing” DNA Strand Patterns on Small Molecules with Control
of Valency, Directionality and Sequence
Tuan Trinh,[a] Daniel Saliba, [a] Chenyi Liao,[b] Donatien de Rochambeau,[a] Alexander Lee Prinzen,[a]
Jianing Li, [b] and Hanadi F. Sleiman*[a]
Abstract: The incorporation of synthetic molecules as corner units in
DNA structures has been of interest over the last two decades. In this
work, we present a facile method to generate branched small
molecule-DNA hybrids with controllable valency, different sequences
and directionalities (5’-3’) using a “printing” process from a simple 3-
way junction structure. We also show that the DNA-imprinted small
molecule can be extended asymmetrically using polymerase chain
reaction and can be replicated chemically. This strategy provides
opportunities to achieve new structural motifs in DNA nanotechnology
and introduce new functionalities to DNA nanostructures.
solid-phase phosphoramidite chemistry, and used them in various
applications. However, the attachment of three or more different
DNA strands on a molecular core requires the laborious synthesis
of molecules with multiple orthogonal protecting groups, followed
by sequential build-up of DNA strands, and suffers from low yield
and tedious purification. Moreover, controlling DNA (5’-3’)
directionality using previous methods is difficult,[16] and is limited
to costly and low-yielding reverse amidites.[4e] To the best of our
knowledge, there is no previous report of a facile synthetic
methodology to covalently link different DNA strands to small
molecules with controllable valency, sequence and directionality.
Our group has recently introduced the concept of DNA
“printing”, which is the covalent transfer of specific patterns of
DNA strands from a DNA nanostructure onto other materials,
such as gold nanoparticles and polymers.[17] Herein, we report a
simple method to covalently “print” different DNA strands from a
self-assembled 3-way junction to a small molecule core using
copper-catalyzed alkyne-azide “click” chemistry (Fig. 1a). DNA-
templated synthesis has evolved considerably as a powerful
method to control and enable reactivity of synthetic molecules.[18]
Our proposed method relies on using a DNA template to ensure
high effective concentration of reactive units in the middle of the
junction, hence facilitating conjugation reactions. As a proof of
concept, we transfer three and four different DNA sequences on
triazide- and tetraazide-functionalized cores, respectively (Fig.
1,2). We demonstrate that the isolated products have exactly pre-
determined numbers and DNA sequences and can be tuned with
respect to DNA directionality and length. Each arm of the
asymmetric branched DNA-small molecule product can be
elongated separately to different lengths in high yield using the
polymerase chain reaction (PCR) (Fig. 4). We then use branched
DNA structures as templates for high-yielding chemical replication
to generate ‘daughter’ branched structures, thus increasing the
scalability of this approach (Fig. 5). Our strategy offers several
advantages compared to the previous methods: 1) It uses
commercially available starting materials and simple,
symmetrically modified small molecule cores, 2) uses end-
modified DNA strands and does not require an in-house DNA
synthesizer, 3) has short reaction time (about 2 hours), 4) is easy
to purify and 5) importantly, it is highly modular, allowing the
incorporation of different DNA sequences with controllable
directionality, length and valency onto different synthetic
molecules.
DNA base-pairing is one of the most reliable and
programmable interactions in nature. These remarkable
properties make this molecule a unique template to finely
organize and control matter at the nanoscale.[1] Most current
approaches, such as DNA tile assembly or DNA origami, use
unmodified DNA strands to guide the assembly and rely on
crossover motifs to create two- and three-dimensional
structures.[2] An attractive complementary approach involves the
use of branched small molecule-DNA hybrids, composed of
multiple DNA strands covalently attached to a small molecule core,
to replace crossover motifs as building blocks for DNA
nanostructures.[3] Using organic vertices with specific geometries
in DNA nanostructures can significantly reduce the number of
strands required, greatly influence assembly outcome, and
increase DNA stability and assembly cooperativity.[3a,4][5]
Moreover, by simply changing the small molecule core in the
building block, new geometries and functionalities can be
added.[6] Branched DNA structures can be used to enhance
hydrogel[7] and nanoparticle formation,[8] DNA metallization,[9]
DNA network formation[10] and DNA crystallization.[11] However,
despite their significant promise, branched DNA hybrid structures
have not been extensively examined as building blocks for DNA
nanotechnology, in comparison to DNA origami or tile assembly.
This is largely due to synthetic challenges in generating these
structures. Branched DNA-small molecules with identical arms
have been generated using solid- or solution-phase synthesis.[3d,
12][13][14]
[3b]
[3d,15]
Over the past years, our group
and others
have
attached two different DNA strands on synthetic vertices using
[a]
[b]
T.Trinh, D. Saliba, D. d. Rochambeau, A. L. Prinzen, Prof. H. F.
Sleiman*
Department of Chemistry
McGill University
801 rue Sherbrooke West, Montreal, QC, Canada H3A 0B8
E-mail: hanadi.sleiman@mcgill.ca
Dr. C. Liao, Prof. J. Li
Deparment of Chemistry
The University of Vermont
Burlington, VT 05405 (USA)
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201809251
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formation of the junction, which is robust enough to not fall apart
at room temperature.
Figure 1. a) Schematic representation of the overall design approach, b)
Chemical structure of reactive strand having 12-carbon organic spacer and
alkyne group and c) 6% native PAGE shows stepwise assembly of the 3WJ.
To examine our hypothesis, we first attempted to “print”
three unique DNA strands (19 bases each) from a designed 3WJ
to a 1,3,5-tris(azidomethyl)benzene core (Fig. 1a). Previously,
this core was used to connect to different DNA strands via solid-
phase synthesis.[3c, 15d] However, instead of multi-step synthesis
of the small molecule core, our method allows simple preparation
of the core as well as the template from commercially available
materials. The middle part is functionalized with 3 reactive 5’-
alkyne-DNA strands, R1, R3 and R5, and 3 rigidifying unmodified
strands, R2, R4 and R6 (Fig. 1). The denaturing gel in Fig. 2a
shows that the trimer with 3 different DNA sequences (R1, R3 and
R5) (3x) was formed with ~ 30% yield after 2 hours, along with
rigidifying strands, scaffold strands (S) and side products (i.e.
monomer and dimer). This represents an average yield of ~ 70%
for each of three “click” reactions on the core. Note that there was
low or no yield of trimer formation in the absence of template
Our strategy uses a self-assembled 3-way junction (3WJ)
as a template (Fig. 1a) containing six scaffold strands S1 to S6 in
addition to single-stranded DNAs (ssDNAs) hybridized to the
middle region of the junction (R1 to R6). Unlike a typical DNA
tile,[19] we break the long middle strand into six individual strands
with three pointing their 5’-end and the other three pointing their
3’-end toward the middle region, in an alternating fashion. In this
region, we hybridize two types of DNA strands: reactive and
rigidifying strands. A reactive strand contains an alkyne functional
group at its end and is separated from the DNA part by a
commercially available hexaethylene (C12) spacer (Fig. 1b),
while the rigidifying strands without functional groups are
hybridized to increase the junction’s geometric definition. Once all
the reactive and rigidifying strands are in pre-designated positions, under the same conditions (Supporting Info-SI-X). To
this region would have high local concentration of the reactive
groups and a small molecule would be able to react and “pick up”
the alkyne-functionalized DNA strands covalently. The resulting
DNA-small molecule product can be then released from the
junction template by denaturation (Fig. 1a). Figure 1c shows a
native polyacrylamide gel electrophoresis (PAGE) as the outcome
of the stepwise 3WJ assembly. Lane 12 shows a near quantitative
characterize the sequence asymmetry of 3x,
3
fully
complementary strands (R1’, R3’ and R5’) which hybridize with
each of the arms of 3x were added sequentially. Fig. 2b reveals
a gradual decrease in mobility of the structures, suggesting
successful hybridization of the individual complementary strands
(see SI-VI for mass spectrometry confirmation).
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decrease in mobility when hybridizing to complementary strands
is shown in the native gel in Fig. 2e, confirming that 4x indeed has
4 different strands grafted on the core. This approach thus offers
full control over the number, sequences, and directionalities of the
DNA strands transferred to the small molecule core.
Figure 3. 3WJ models from MD simulations with 3 different reactive strands in
the center with zoom-in top/side views on the right.
We were then interested in studying the effect of linker
length, connecting the DNA portion to the alkyne moiety, on the
efficacy of the transfer process. Interestingly, we observed that
the trimer formation efficiency significantly decreased to 7% and
17%, by shortening the 12-carbon linker to 3-carbon and 6-carbon,
respectively. When we completely removed the organic spacer
and connected the DNA strand directly to the alkyne monomer,
there was no trimer formation (see SI-XIV). We hypothesized that
the length of the spacer plays a critical role in bringing the alkyne
groups near the azides on the small molecule. Molecular
dynamics (MD) simulations of the 3WJ (Fig. 3) were performed to
gain insight into the molecular level. To understand the regulation
of the reactive strands in the junction to the final small-molecule
products, we simulated the 3WJ with three reactive-strand
models: 1) the C12 spacer (~18 Å) connecting the DNA and the
alkyne; 2) without the C12 linker; 3) a longer spacer of 5 thymine
bases (~24.5 Å). Simulations show that the spacer length indeed
affects the inter-reactive strand distance towards the product yield.
Within 30 ns, there is at least one pair of reactive strands within
~15 Å measured by nitrogen-nitrogen distance (in alkyne
modifier) in the C12 spacer system (SI-XVI). In contrast, in the no-
C12 spacer system, no reactive strands were found to maintain
within 15 Å. When we replaced the C12 spacer with a longer 5
thymidine spacer, three reactive strands were able to reside
closely together within ~15 Å. Thus, we predicted a spacer with
length ~18 Å is essential to yield desired products. In some cases,
having a long organic linker is not highly desirable (e.g. in terms
of flexibility and hydrophobicity). Based on our simulations, we
Figure 2. a) 15% denaturing PAGE for trimer formation. Lane 1: crude reaction
mixture between fully assembled 3WJ with triazide molecules. Lane 2: fully
assembled 3WJ in denaturing condition. b) Stepwise addition of complementary
strands to 3x c) Schematic representation and native PAGE of the
addressability of the 3x. d) Stepwise addition of complementary strands to 4x.
We were interested in changing the directionality (5’-3’) of the
transferred DNA strands onto the core. The 3WJ allows us to
easily switch the reactive alkynes to the 3’ ends of the 3 other
strands (R2, R4 and R6), pointing toward the core of the 3WJ. We
obtained a similar yield of the reverse direction trimer (with the 3’-
ends of the DNA strands connected to the small molecule core)
that would be difficult to achieve using previously reported
methods (SI-XI).[13-14,15d,16] Moreover, we were able to covalently
link three DNA strands with different lengths (19-, 36- and 41-mer)
to the benzene core (SI-XII).
To broaden the scope of this approach, we examined the
transfer of 4 unique DNA strands onto a flexible tetraazide-
functionalized molecule (Fig. 2c). We hybridized 4 reactive
strands (3 of them with 5’-alkynes and 1 with 3’-alkyne) and 2
rigidifying strands to the 3WJ (Fig. 2c). The denaturing gel in Fig.
2d reveals the formation of the tetramer product (4x) with a yield
of 26% (~70% yield for each reaction). Similarly, the sequential
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experimentally examined trimer formation by directly linking
(e.g. quantum dots and/or gold nanoparticles). To probe the utility
alkyne-functionalized DNA (no organic spacer) to the organic core. of our method, we set out to elongate 2 arms (R1 and R3) of the
We substituted the C12 spacer with a 5-thymidine spacer and we
observed formation of the trimer with ~35% yield, which is slightly
improved compared to that using 12-carbon linker (SI-XIV).
These results demonstrate that the spacer length is crucial for the
process, and that direct linking of DNA to the small molecule core
is efficient.
asymmetric trimer simultaneously to different lengths using PCR.
Since our method enables full control over sequence and length
of the DNA strands grafted on the small molecule, we were able
to synthesize a benzene core (called trimer-2primers – Fig. 4a)
connected to 3 different DNA arms: a 41-nucleotide (nt) arm
containing a forward primer 1 (R1-primer1), a 36-nt arm
containing a forward primer 2 (R3-primer2) and a 19-nt arm (R5).
Two different long double-stranded DNA (dsDNA) were used as
templates. One of the sequences was generated using ‘temporal
growth’, a process previously developed by our lab, while the
other was made using standard phosphoramidite DNA
synthesis.[20a] Lane 7 in the agarose gel (Fig. 4c) revealed the
formation of the PCR desired product with a yield of 70-80% (3
repeats). Then, the band of interest was excised and further
characterized by atomic force microscopy (AFM) under dry
conditions. For easier visualization, we hybridized the third arm
with an 82 bases dsDNA. Unambiguously, AFM confirmed that
the product has exactly 3 arms, each with a different length (Fig.
4b). The longest arm was measured to be 119 ± 9 nm (N=100),
which is in accordance with the expected length of 121 nm. The
two other arms were 46 ± 5 nm (N=100, theoretical length for 132
bp dsDNA ~ 45 nm) and 28 ± 4 nm (N=100, theoretical length for
85 bp dsDNA ~ 29 nm), respectively (see SI-XV for gel
characterization). This is the first time a branched DNA-small
molecule motif has been elongated asymmetrically using PCR.
DNA is one of the most classical examples of self-replication
- the ability to make its own copies in nature. This concept has
been recently transferred from living cells to DNA nanotechnology
to replicate DNA tiles and DNA origami rafts.[22] Working towards
this path may solve the problem of DNA structure scalability.
Therefore, we would like to see if our asymmetric branched trimer
structure (trimer-2primers in Fig. 4) can serve as a mother
template to produce daughter generations chemically. Previously,
von Kiedrowski et al. introduced this concept using a symmetrical
junction.[23] First, we hybridized alkyne-functionalized T2-
R1comp, T3-R3comp and T4-R5comp to the ‘mother’ template
(trimer-2primers), followed by the addition of the triazide
molecule. After denaturation, the ‘daughter’ structure,
characterized by a higher gel mobility shift, was formed (Fig. 5b-
lane 1), and the biotinylated ‘mother’ template was recovered
using streptavidin-functionalized magnetic beads. The product
appeared as a tight band by gel electrophoresis, suggesting that
a single trimer was formed with a yield exceeding 90% (Fig. 5b
lane 3). Control experiments without template under the same
conditions showed no branched trimer formation (Fig. 5b – lane
2). In addition, after 3 rounds of replication (Fig. 5a), the daughter
was amplified 3 times more than in a single cycle (SI-XVII). Our
future work will focus on the exponential growth of both ‘mother’
and ‘daughter’ structures.
Figure 4. a) Scheme for DNA arm elongation using PCR. b) AFM images (in
dry condition) confirm that the elongated trimer structure has exactly 3 arms of
different lengths. c) 2% agarose gel in TAE buffer confirms the elongation of the
DNA trimer structure. Lane 1: trimer-2primers (control); Lane 2: PCR product
of ssDNA (113 bases); Lane 3: PCR product of ssDNA (336 bases); Lane 4:
PCR products of both ssDNA (113 bases) and ssDNA (336 bases) in the same
mixture; Lane 5: Elongation of 1 arm in trimer to 132 bases in length; Lane 6:
Elongation of 1 arm in trimer to 355 bases in length; Lane 7: Elongation of 2
arms simultaneously in trimer.
Long strands of DNA with controlled sequences are of
interest for many applications including data storage, material
organization and molecular electronics.[20] However, only up to
200 bases ssDNA can be practically obtained using conventional
solid-phase synthesis. Therefore, simply hybridizing the 3-arm
template with a long DNA strand will be limited by the length of
ssDNAs made by a DNA synthesizer. Moreover, the stability of
the construct would be highly dependent on a short hybridization
region with the 3-arm template. One reliable method to elongate
a DNA strand is PCR. Previously, Bao et al. demonstrated
extension of identical DNA arms to micron-sized structures from
small molecule cores, but the asymmetrical elongation of DNA
arms is still a challenge.[21] Asymmetric DNA elongation can
significantly enhance the complexity and information content of a
DNA building block, that can potentially serve as a unique scaffold
for DNA metalation and for organizing precisely different materials
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We thank the Natural Sciences and Engineering Research
Council of Canada (NSERC), the Canadian Institutes for Health
Research (CIHR), and the Canada Research Chairs Program for
financial support. We also thank Dr. Pongphak Chidchob for
helpful discussions. H.F.S. is a Cottrell Scholar of the Research
Corporation.
Keywords: DNA nanotechnology • DNA printing • DNA-
templated reactions• Self-assembly • Branched DNA structures
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Acknowledgements
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10.1002/anie.201809251
Angewandte Chemie International Edition
COMMUNICATION
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COMMUNICATION
Breaking the symmetry of small
molecules using DNAs: A highly
modular method to covalently
transfer different DNA sequences
with controllable directionality,
length and valency onto different
synthetic molecules is reported.
These DNA-imprinted small
molecules can be elongated
asymmetrically to different length in
high yield using polymerase chain
reaction and can be chemically
replicated.
Tuan Trinh, Daniel Saliba, Chenyi
Liao, Donatien de Rochambeau,
Alexander Lee Prinzen, Jianing Li,
Hanadi F. Sleiman*
Page No. – Page No.
“Printing” DNA Strand Patterns on
Small Molecules with Control of
Valency, Directionality and Sequence
This article is protected by copyright. All rights reserved.