Chem148 Winter 2021 Reading assignment 1 Name: Amy Nguyen

Read carefully the article by Gerling et al (2015) “Dynamic DNA devices and assemblies formed by
shape-complementary, non-base pairing 3D components” and answer to the following questions.
P.S: Note that the Supplementary Materials for the article has been posted on GauchoSpace. Provide
your answers after each question on the following printout sheet.

(1) What is the source of inspiration for this work? Briefly describe in one sentence.

Although rare, weaker binding interactions than base pairing exist for nucleic acids. Specifically
speaking, an RNA-based enzyme known as ribonuclease P or RNase P cleaves its substrate, pre-transfer
RNA (tRNA), at the 5’ leader strand to produce mature tRNA. By mimicking the way in which RNase P
recognizes tRNA, discrete conformations can be produced with this method of programmable self-
assembly.

(2) In two to three sentences, explain what are the main achievements of this work…

By influencing DNA self-assembly through cation concentration, temperature, or an allosteric

mechanism, the production of an actuator, a switchable gear, an unfoldable nanobook, and a nanorobot
was achieved. To stabilize these homo- and hetero-multimeric objects as well as the reconfigurable
devices, the bonding energy produced from the stacking of nucleobases would compete against the
electrostatic repulsion produced from the same phenomenon. These multidomain assemblies were then
visualized via imaging by electron microscopy, fluorescence resonance energy transfer spectroscopy
(FRET), and electrophoretic mobility analysis.

(3) Briefly describe the principle of DNA origami used to create the synthetic DNA objects?

In order to bind to other molecules, proteins are frequently required to find interfaces that are compatible
with the shape of the protein. Consequently, these fragile interactions allow for turnover and
conformational dynamics to occur. In essence, DNA origami consists of arranging building blocks into
certain sequences while maintaining the structure of higher-order complexes through weak spatial
interactions, resulting in the ability to form synthetic DNA objects.

(4) How are the DNA objects assembled to one another? At a structural level, what are the types of
interactions used to promote assembly? Explain how it is possible to program specific assembly at
the level of the DNA?

There are three means of changing DNA self-assembly: (1) changing the concentration of cations; (2)
changing the temperature of the solution; and (3) an allosteric mechanism (i.e. a toehold-mediated strand
displacement reaction). Using this technique, encoding specific conformations at this level of DNA is
merely a matter of simple geometrical considerations (i.e. the stacking of the nucleobases) and is not as
complex of an ordeal as programming intricate strand sequences.

(5) How did the authors demonstrate that they form the correct assemblies?

These multidomain assemblies were visualized via imaging by negative-stain electron microscopy,
electrophoretic mobility analysis, and ensemble and single-molecule fluorescence resonance energy
transfer spectroscopy (FRET).
Chem148 Winter 2021 Reading assignment 1 Name: Amy Nguyen

(6) How did the authors obtained the DNA object shown in Figures 1E,F? What is the trigger for
switching its shape?

A switchlike DNA object was obtained by connecting two rigid beams with a pivot, where the switch
can exist in an ensemble of open states or in a closed state. When the two beams come together, the
shape-complementary regions of the double-helical DNA domains click into each other and form a
closed state structure. The switch is triggered by increasing the cation concentration (i.e. from 5 mM
MgCl2 to 25 mM MgCl2), thereby entering a closed state.

(7) What is Figure 2 about? Briefly explain how the authors are able to control the behavior of the
switching object.

Figure 2 discussed the effects of changing cation concentration or temperature and using a site-specific
allosteric mechanism on shape-complementary DNA objects. By increasing the cation concentration, the
conformational equilibrium shifted from open states to closed states for the switch and dimeric bricks.
Through cation titration, they were able to plot the two states on a free-energy diagram, revealing that
reversible equilibrium was linearly dependent on the concentration of cations.

(8) Briefly explain Figure 2B by emphasizing the structural differences between the various DNA
constructs tested. What is the most stable assembly strategy?

Four variants were studied under ensemble FRET by titrating MgCl2 with solutions containing these
switch variants: a variant with three-base-long hybridization bonds rather than 16 stacking bond sites; a
variant with 16 possible stacking bonds in the closed state; a variant with 12 possible stacking bonds in
the closed state; and a variant that cannot form stacking bonds to stabilize the closed state. As the only
variant that had an observable transition over a narrow concentration interval, the switch version with 16
stacking bonds was the most stable assembly strategy; the others were unable to switch to the open state
under low cation concentration conditions without compromising the stability of the switch itself.

At a DNA structural level, what is necessary for reversible switching as shown in Figure 2C?

To reversibly shift the equilibrium from closed or dimeric states to open or monomeric states, the
temperature of the solutions containing dimeric bricks or switch variants would be changed.

(9) What is the technique used to follow the behavior of the switch object? Explain how the DNA
objects are labeled with dyes.

Organic dyes Atto550 and Atto647n were used to obtain ensemble and single-molecule fluorescence
resonance energy transfer (FRET) spectra of the switch objects. Furthermore, TEM imaging and
electrophoretic mobility analysis were taken as cation concentration and temperature were varied for the
switch object.

(10) Explain how it is possible to control reversibly the behavior of the object with DNA
oligonucleotides, “A*t” and “At” as shown Figures 2E,F. What is the purpose of “A*t”? How is
Chem148 Winter 2021 Reading assignment 1 Name: Amy Nguyen

“At” able to reverse the process? Provide a schematic that describes how the “toehold” mechanism is
used in this specific case.

Akin to At, A*t is composed of DNA single strands and is complementary to single-stranded loops on
the switch. Due to their similarity, At can readily displace A*t from the switch in a toehold-mediated
displacement reaction. Since DNA origami objects are malleable, their binding equilibrium can be
affected allosterically, such as through deformations on binding sites. Hybridization of these deformed,
loop-complementary DNA single strands can lead to inhibition of attractive interactions. By displacing
A*t and thus essentially removing this single strand from the loops, reversal of the deformation and
restoration of the attractive interactions could be achieved.

(11) What are the different applications proposed in this paper? In few words, state each of them and
indicate where the results are display on Figures 3 and 4.

Self-complementary bricks were able to self-assemble into homomultimeric filaments via sufficient
rigidity induced by constraining the position of long-range binding partners (Figs. 3A and 3B, left).
Changing cation concentration allows for the shrinkage and growth of these filaments (Fig. 3B, right),
which is significant to polymerization-based propulsion applications. Specifically, a hexagonal
multilayer DNA origami brick can be formed from a monomer (Fig. 3C) and then self-assembled into
2D hexagonal lattices by increasing the concentration of cations (Fig. 3D). By altering the shape-
complementary stacking interactions of the hexagon brick, the hexagon bricks could then self-assemble
into two-stranded filaments (Fig. 3E), which could be studied for its nucleated growth. Though this
study was performed on the nanoscopic level, the scale can be enlargened to a micrometric perspective,
allowing for the synthetic assemblies of active biological components such as in muscle tissue (Fig. 3F);
reversible expansion and contraction are influence by the decrease and increase of cations in solution.

Using the shape recognition method, a variety of reversibly reconfigurable DNA devices could be self-
assembled, such as reconfigurable gear that can be switched between a pinionrack-like shape and a gear
like shape (Fig. 4A), a reconfigurable nanobook that can reversibly fold and unfold (Fig. 4B), a
heterotrimeric complex consisting of three asymmetric subunits that assemble based on shape
recognition (Fig. 4C), and a nanorobot with various stacking bonds that allow it to perform several
actions (Fig. 4C). Simply considering geometry in 3D space could allow a designer to encode complex
higher-order objects with minimal nucleobase stacking interactions, as seen with the multilayer DNA
origami objects in honeycomb-lattice and square-lattice packing (Fig. 4D).

(12) In Figure 4, how is the behavior of the nano-objects controlled?

The creation of the reconfigurable gear is made possible by adjusting the cation concentration (e.g. the
amount of MgCl2). Increasing and decreasing the concentration operates as the “switch” of the gear,
causing the cation-dependent surface to open and close.

(13) To your opinion, do you think that the results presented in this paper are significant? Briefly
justify your answer.

The implications of this shape-co opens new doorways for DNA-based nanotechnology. As mentioned
in the “Applications” portion of the paper, these results can lead to profound biotechnological
Chem148 Winter 2021 Reading assignment 1 Name: Amy Nguyen

innovations within the fields of “therapeutics, biosensing, and active plasmonics, and responsive
nanostructured materials.” The simplicity of this method can allow us to readily observe the behavior of
DNA self-assembly without much difficulty (i.e. generating detailed strands of DNA sequences).