Developmental Self-Assembly of a DNA Ring with Stimulus-
Responsive Size and Growth Direction
Allison T. Glynn,
¶
Samuel R. Davidson,
¶
and Lulu Qian
*
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ABSTRACT:
Developmental self-assembly of DNA nanostructures
provides an ideal platform for studying the power and programm-
ability of kinetically controlled structural growth in engineered
molecular systems. Triggered initiation and designated sequencing
of assembly and disassembly steps have been demonstrated in
structures with branches and loops. Here we introduce a new strategy
for selectively activating distinct subroutines in a developmental self-
assembly program, allowing structures with distinct properties to be
created in response to various molecular signals. We demonstrate this
strategy in triggered self-assembly of a DNA ring, the size and growth
direction of which are responsive to a key molecule. We articulate that
reversible assembly steps with slow kinetics at appropriate locations in
a reaction pathway could enable multiple populations of structures with stimulus-responsive properties to be simultaneously created
in one developmental program. These results open up a broad design space for the self-assembly of molecules with adaptive
behaviors toward advanced control in synthetic materials and molecular motors.
■
INTRODUCTION
Molecular self-assembly is key to the functionality of living
cells, allowing lipids, nucleic acids, and proteins to organize
themselves into structures with desired shapes and properties.
Understanding the principles of self-assembly in engineered
molecular systems is fundamentally important to control the
behavior of biomolecules for technological advances. DNA
self-assembly is one of the most well-studied areas of
engineered molecular self-assembly.
1
Complex shapes with
up to gigadalton sizes have been created with nanometer
precision.
2
−
5
Moreover, a self-assembly process could be
designed to carry out complex computation and algorithms.
6
,
7
Most DNA self-assembly processes investigated so far take
place spontaneously during thermal annealing, but some
exhibit isothermal behavior in response to a triggering
signal.
8
−
10
Triggered self-assembly processes allow desired
structures to grow at desired times, while the isothermal
property allows for applications where temperature changes are
undesired, for example, in a biological environment.
11
,
12
Similar to how the kinetics of growth in multicellular
development is orchestrated by genetic programs, the kinetic
pathway of triggered self-assembly can be controlled by
molecular programs encoded in DNA; this type of behavior
has been referred to as developmental self-assembly.
13
The
kinetic control was achieved by toehold-mediated DNA strand
displacement,
14
where the reaction of an initiator strand with a
hairpin motif by toehold binding and branch migration reveals
a previously sequestered toehold for subsequent reactions.
Using this mechanism, dendritic structures
9
and a tetrahe-
dron
13
were created with prescribed sequences for every self-
assembly step.
The prior investigations raised an important challenge
regarding the design space of triggered self-assembly. As seen
in biological systems, development can be in
fl
uenced by
changing environmental conditions throughout the entire
growth process rather than just within the initiation step.
What new design principles can be established to enable the
self-assembly of DNA nanostructures with stimuli-responsive-
ness more deeply embedded within the growth process? To
begin answering this question, here we show that distinct signal
molecules can be designed to selectively activate a subroutine
(e.g., a subset of steps) in a developmental self-assembly
program, resulting in the assembly of structures of varying sizes
or growth directions from the same set of building blocks. In
these examples, the signal molecules encode both the start and
stop conditions of a growth process, paving the way for future
explorations involving more complex conditions.
Various strategies for growing DNA nanostructures with
programmable sizes have been developed. For example,
increased numbers of unique strands were used to create
Received:
April 10, 2022
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DNA tubes with increased circumferences,
15
and distinct
connector strands with a speci
fi
co
ff
set were used to enforce
how wide a sheet must grow before it rolled up into a tube.
16
Due to the nature of spontaneous self-assembly, these
strategies lead to the immediate growth of partial target
structures once the DNA strands are mixed together (
Figure
1
a). Seeded growth is possible in tile self-assembly systems,
which allow the width of DNA ribbons to be controlled using a
DNA origami structure as an information-bearing seed.
17
In
that case, nucleation only occurs when the seed is present.
However, once the growth begins, DNA tiles will sponta-
neously bind to each other, as by default they are all activated.
By contrast, developmental self-assembly utilizes hairpin motifs
that are activated one at a time. This unique property makes it
possible to design a system where the entire growth process is
inhibited through kinetic traps, parts of which can be
selectively activated upon speci
fi
c signals. These signals could
then alter the outcome of self-assembly (
Figure 1
b).
■
RESULTS AND DISCUSSION
To demonstrate the concept of developmental self-assembly
with stimulus-responsive properties, we designed a set of
hairpins that could be triggered to form DNA rings with
varying sizes depending on the identity of a key that functioned
as both an initiator and a terminator (
Figure 2
a). As shown in
the abstract reaction graph (notation explained in the
fi
gure
caption), a unique toehold composing the output port in each
of the hairpins is initially sequestered, preventing the hairpins
from interacting with each other when all keys are absent. A
key reacts speci
fi
cally with one of the hairpins, activating its
output port for the next assembly step. A cascade of assembly
reactions occurs until the activation of a hairpin whose output
port matches the input port on the key. A disassembly reaction
then takes place to release a strand from the key and close the
ring, completing the self-assembly pathway. As shown in
Figure
2
b, each of the the seven unique hairpins consists of two
exposed toeholds (input ports), a common branch migration
domain (colored in black), and a sequestered toehold (output
port). Each of the three unique key molecules consists of two
strands, one of which opens up the
fi
rst hairpin and the other
of which will be released by the last hairpin in the designated
self-assembly pathway. The released strand is labeled with a
fl
uorophore to detect pathway completion.
Each assembly step that opens up a hairpin is a reversible
strand-displacement reaction (
Figure 3
). The forward reaction
is driven by additional base pairs in the toehold domain, while
the backward reaction is driven by the entropic gain of one free
molecule. Toehold sequences were designed in NUPACK
18
to
ensure approximately equal forward and backward reaction
rates based on the equilibrium constant estimated with 100 nM
reactant concentrations. The
fi
nal disassembly step that closes
the ring is an irreversible strand-displacement reaction driven
forward by entropy and possibly additional base pairs. A short
loop domain in each hairpin provides the desired structural
fl
exibility for ring formation. The loop domain in the last
Figure 1.
Concept of (a) spontaneous and (b) developmental self-
assembly, which create DNA rings with distinct sizes.
Figure 2.
Design of a DNA ring with a stimulus-responsive size. (a)
Abstract reaction graph. Each hairpin is represented as a node with
three ports, and each key is represented as a node with two ports.
Triangles and circles indicate input and output ports, respectively,
while their open or
fi
lled representation corresponds to an exposed or
sequestered toehold. Solid and dashed black arrows indicate assembly
and disassembly reactions, respectively. Gray arrows indicate possible
reactions that are not used in the designed ring formation. (b)
Domain-level strand diagrams. Each unique toehold and branch
migration domain is labeled with a distinct letter. Asterisks indicate
sequence complementary. The three keys that trigger the self-
assembly of DNA rings with four, six, and eight strands are labeled as
key4, key6, and key8, respectively.
Figure 3.
Reaction pathway of the formation of a four-stranded ring.
Forward and backward reactions are indicated by
fi
lled and open
arrowheads, respectively. Each toehold, branch migration, and loop
domain has seven, six, and four nucleotides, respectively.
Journal of the American Chemical Society
pubs.acs.org/JACS
Article
https://doi.org/10.1021/jacs.2c03853
J.Am.Chem.Soc.
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−
XXX
B