Chem148

Reading assignment 1 Name:

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.
This work is inspired by the way RNA molecules interact with their substrates, in this case Rnase P RNA
interacting with the tRNA. RNase P recognizes the shape rather than the specific sequence of tRNAs.

(2) In two to three sentences, explain what are the main achievements of this work…
In this study, the authors use programmable self-assembly with DNA to produce discrete, shape-
complementary three-dimensional (3D) components that interact via short-ranged, nucleobase stacking
bonds. They present three means for actively influencing the conformation of objects once assembled: (i)
changing cation concentration; (ii) changing solution temperature; and (iii) a site-directed allosteric
mechanism based on toehold-mediated strand displacement reactions. The method developed by the
authors allows a designer to encode a diversity of readily reconfigurable DNA devices and assemblies
based on simple geometrical considerations and without having to program detailed strand sequences
for connecting components.

(3) Briefly describe the principle of DNA origami used to create the synthetic DNA objects?
DNA origami typically refers to a DNA self-assembly technique where a long single stranded molecule,
acting as a “scaffold”, is folded into an arbitrary 2D or 3D shape in presence of small oligonucleotides
acting as staples. It is essentially based on base pair specificity.

See posted paper by Rothemund (2006) Nature

(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?
In this study, once the DNA objects are built using DNA origami, they are assembled to one another by
shape complementarity. Nucleobase stacking interactions engage at the double-helical interfaces of the
shape-complementary protrusions and recessions when two interacting DNA objects are brought into
contact, but only upon correct fit of the helices and given correct helical orientation of the interfacial
nucleobase pairs.

(5) How did the authors demonstrate that they form the correct assemblies?
They use multiple experimental techniques: analysis and purification of the DNA objects by gel
electrophoresis, visualization by Transmission Electron Microscopy with negative staining, ensemble
and single-molecule fluorescence resonance energy transfer (FRET) techniques.

(6) How did the authors obtained the DNA object shown in Figures 1E,F? What is the trigger for
switching its shape?
They used DNA origami to generate a multidomain DNA object made up of two rigid beams connected
by a pivot. The trigger is the magnesium concentration.

Chem148 Reading assignment 1 Name:

(7) What is Figure 2 about? Briefly explain how the authors are able to control the behavior of the
switching object.
Reversible reconfiguration of shape-complementary DNA objects by changes in cation concentration
or temperature and with a site-specific allosteric control mechanism.

(8) Briefly explain Figure 2B by emphasizing the structural differences between the various DNA
constructs tested. What is the most stable assembly strategy?
Four different structures are tested: a switch object with complementary hybridization bonds (between
complementary DNA tails) (triangle), another with 16 stacking bonds (circle), another with 12 stacking
bonds (diamond) and a variant unable to stabilize its closed conformation (hexagon).
The most stable DNA object in the close conformation is the one with hybridization bonds. However, for
reversibility, the assemblies with 12 or 16 stacking sites have the best ability to fluctuate between the
two conformations when heated or salted.
As expected, the structure without bonds is unable to close.

At a DNA structural level, what is necessary for reversible switching as shown in Figure 2C?
For reversible switching in function of the temperature, it is necessary to be within a difference of free
energy between the close and open state that is not too far from zero kcal/mol in the salt condition
tested. At 10 mM MgCl2, the stacking energy of the switch object with 16 stacking bonds are in a regime
allowing rapid change of equilibrium in function of temperature, something that is not possible for the
more stable switch object with hybridization bonds.

(9) What is the technique used to follow the behavior of the switch object? Explain how the DNA
objects are labeled with dyes.
The techniques are ensemble and single-molecule fluorescence resonance energy transfer (FRET). The
dyes used in FRET consist of two fluorophores, a donor (Atto647) and an acceptor (Atto550), which are
linked to oligonucleotides. Each fluorophore-oligonucleotide is complementary to a different region of
the DNA 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 “At”
able to reverse the process? Provide a schematic that describes how the “toehold” mechanism is used in
this specific case.
The switch object has a flexible single stranded loop region “A” that could eventually be paired to
“A*t”. When “A*t” is present, it assembles with the single stranded loop on the switch to form a rigid
DNA helical duplex that disturbs one of the shape complementary stacking interface, leading to the
opening of the switch. Because “A*t” features an additional nine-bases-long toehold motif, it can be
peeled off from the loop region of the switch by the “At” olignucleotide that is fully complementary to
“A*t”. A better schematic is the one
from Figure S30 of the SOM and the
one from the website:
https://en.wikipedia.org/wiki/Toehol
d_mediated_strand_displacement.

Chem148 Reading assignment 1 Name:

(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.
First, the position of binding partners may be constrained with sufficient rigidity to template long-range
orientation with apparently seamless integration of up to hundreds of monomers and absence of bending
deformations up to the micrometer length scale (Shown Figure 3A).
Second, the ability to reversibly shrink and grow filaments is of interest for creating active materials
and in polymerization-based propulsion applications (Shown Figure 3B).
Third, different higher-order objects may be created from a family of similar building blocks by using
simple shape alterations and without having to design interfaces with specific DNA sequences (Shown
Figure 3C,D,E).
Fourth, nanoscopic changes in the geometry of individual building blocks may also be amplified up to
the micrometer scale, which is an important step in creating synthetic assemblies that could attain the
high level of structural integration of active components seen in biology, such as in muscle tissue
(Shown Figure 3F).
Fifth, various reversibly reconfigurable DNA devices with arbitrary shapes can also be engineered
(Shown in Figure 4).

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

(13) To your opinion, do you think that the results presented in this paper are significant? Briefly justify
your answer.
The results presented are clearly increasing the toolbox of DNA origami to generate interesting
responsive nano-objects. The real usefulness of the described objects is certainly questionable but the
paper nicely re-emphasizes the potential of the DNA origami technique for controlling matter at the
nanoscale.