A report about Biochemistry article.

Hello,Thank you for taking the time to read my post.I need to make a report about a Biochemistry article ( Challenges and opportunities for structural DNA nanotechnology) The report must include the answers for these questionsWhat is “structural DNA nanotechnology”? List three technique challenges in the field of structural DNA nanotechnology from the review article that you found interesting (or important, hard to overcome).Comment on at least one of the future applications for structural DNA nanotechnology discussed in this review article. Conclude with your thoughts on this review article.

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PUBLISHED ONLINE: 6 NOVEMBER 2011 | DOI: 10.1038/NNANO.2011.187
Challenges and opportunities for structural
DNA nanotechnology
Andre V. Pinheiro1, Dongran Han1,2, William M. Shih3,4,5* and Hao Yan1,2*
DNA molecules have been used to build a variety of nanoscale structures and devices over the past 30 years, and potential
applications have begun to emerge. But the development of more advanced structures and applications will require a number of
issues to be addressed, the most significant of which are the high cost of DNA and the high error rate of self-assembly. Here we
examine the technical challenges in the field of structural DNA nanotechnology and outline some of the promising applications
that could be developed if these hurdles can be overcome. In particular, we highlight the potential use of DNA nanostructures in
molecular and cellular biophysics, as biomimetic systems, in energy transfer and photonics, and in diagnostics and therapeutics
for human health.
he field of structural DNA nanotechnology can be traced back
to the words written by Nadrian Seeman in 1982: “It is possible to generate sequences of oligomeric nucleic acids which
will preferentially associate to form migrationally immobile junctions, rather than linear duplexes, as they usually do.”1 Seeman had
wanted to organize proteins in three-dimensional (3D) crystals so
that he could study their structure with X-ray crystallography. Three
decades later the field has outgrown its roots in protein crystallography and delivered numerous advances in the control of matter on
the nanoscale (Fig. 1). The history and state of the art in structural
DNA nanotechnology have been widely reviewed2–7. Here, instead,
we seek to stimulate discussions about the future of the field.
Research in structural DNA nanotechnology began with the
construction of relatively flexible branched junction structures8
and topological structures9–16, progressing to the fabrication of
crossover DNA tiles with greater rigidity. These tiles could be used
to assemble higher-order periodic and aperiodic lattices17–30, and
nanotubes31–37. A landmark of periodic DNA structure assembly
was achieved by Seeman and co-workers38 in 2009 with the formation of 3D DNA crystals from tensegrity triangles39 that diffract
X-rays to 4 Å resolution.
One of the most important development in structural DNA
nanotechnology since the introduction of the crossover tile has
been the use of a ‘scaffold’ DNA strand for the assembly of aperiodic structures. It had been previously demonstrated that a long
single-stranded DNA chain could be used to organize doublecrossover tiles into barcode-patterned lattices40, and that a 1.7-kb
single-stranded DNA chain could serve as a scaffold for the assembly of a 3D wire-frame octahedron41. The breakthrough came with
the concept of ‘DNA origami’, where a long scaffold strand (singlestranded DNA from the M13 phage genome, ~7,429 nucleotides
long) was folded with the help of hundreds of short ‘staple’ strands
into defined two-dimensional (2D) shapes42. The scaffold is thought
to corral the component strands in a way that leads to high effective
concentrations and proper stoichiometry, so that even unpurified
oligonucleotides can be used to produce well-formed 2D structures
in near-quantitative yields. DNA origami structures can also be
used as molecular pegboards with a resolution of 4–6 nm, and they
have been widely used in the assembly of heteroelements such as
proteins and nanoparticles (see below).
Three general strategies have been explored to extend DNAorigami nanoconstruction to the third dimension. The first relies
on folding interconnected individual or continuous DNA origami
sheets into hollow 3D cages43–46. The second method builds custom 3D shapes by constraining layers of helices to a honeycomb47
or square lattice48; the targeted insertion and deletion of base pairs
within such rigid 3D blocks allows twisted and curved 3D objects
to be made49. The third strategy is to stack concentric double-helical
circles containing different numbers of turns, and therefore having
differing circumferences, to match the rounded contours of a target
container shape50.
Taking advantage of the sequence specificity and the resulting
spatial addressability of DNA nanostructures, many of the DNA
nanoarchitectures listed above have been used for the organization of heteroelements such as proteins22,29,51–57, peptides58, virus
capsids59, nanoparticles60–74 and carbon nanotubes75. And in turn,
several of these DNA-directed assemblies have led to unique and
improved functional properties, such as increased enzyme-cascade
activities76,77 due to spatially positioned enzyme pairs, and shifts of
surface plasmon resonance controlled by custom arrangement of
nanoparticles78–80 through DNA-mediated self-assembly.
Nearly 30 years after Seeman’s original proposal, scientists now
have at their disposal a multitude of designs and techniques with
which to devise increasingly complex systems for scientific and technological applications. However, structural DNA nanotechnology is
still at a relatively early stage in its development, and here we will
discuss the challenges that must be overcome to reach greater levels
of control and functionality.
Technical challenges
DNA origami has already provided a spectacular example of the
power of static self-assembly as a design paradigm to create custom
cookie-cutter shapes each with a mass of ~5 megadaltons (which is
twice the mass of a ribosome). Could much more complex DNAnanostructure designs be made in the future? It is instructive to note
that the number of transistors per integrated circuit has doubled
Center for Single Molecule Biophysics, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA, 2Department of Chemistry and
Biochemistry, Arizona State University, Tempe, Arizona 85287, USA, 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts 02115, USA, 4Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA, 5Wyss Institute
for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02138, USA. *e-mail: hao.yan@asu.edu; William_Shih@dfci.harvard.edu
NATURE NANOTECHNOLOGY | VOL 6 | DECEMBER 2011 | www.nature.com/naturenanotechnology
© 2011 Macmillan Publishers Limited. All rights reserved
DNA tile
DNA directed
20 nm
20 nm
75 nm
200 nm
50 nm
Figure 1 | Examples of structural DNA nanotechnology. a, Seeman’s original proposal consisted of using immobile DNA junctions (left) to build 3D
scaffolds that could be used to organize proteins (right)1. b, Important milestones in structural DNA nanotechnology: the first wireframe 3D cube10 (left),
DNA origami (centre) and a 3D periodic structure composed of tensegrity triangles (right). c, DNA periodic arrays composed of double-crossover tiles
(left), 4 × 4 tiles (centre left), three-point star tiles (centre right) and double-crossover-tile-based algorithmic assembly of Sierpinski triangles (right).
d, Three-dimensional DNA origami: a hollow box (left pair of images), a multi-layer square nut (centre left pair), a square-toothed gear (centre right pair)
and a nanoflask (right pair). e, DNA nanostructure-directed patterning of heteroelements: double-crossover tiles for the organization of gold nanoparticle
arrays (left), DNA origami for the assembly of carbon nanotubes (centre left), biotin–streptavidin protein patterning of 4 × 4 tiles (centre), aptamerdirected assembly of thrombin arrays on triple crossover tiles (centre right), and Snap-tag and His-tag mediated orthogonal decoration of DNA origami
(right). Figures reproduced with permission from: b, Nadrian C. Seeman (left), ref. 42, © 2006 NPG (centre), ref. 38, © 2009 NPG (right); c, ref. 19, © 1998
NPG (left), ref. 22, © 2003 AAAS (centre left), ref. 24, © 2005 ACS (centre right), ref. 30, courtesy of P. Rothemund (right); d, ref. 43, © 2009 NPG (left),
ref. 47, © 2009 NPG (centre left), ref. 49, © 2009 AAAS (centre right), ref. 50, © 2011 AAAS (right); e, ref. 62,© 2004 ACS (left), ref. 75, © 2010 NPG
(centre left), ref. 22, © 2003 NPG (centre), ref. 53, © 2005 Wiley (centre right), ref. 57, © 2010 Wiley (right).
every two years for the past four decades — roughly a one-millionfold increase between 1971 and 2011. Such an increase in complexity
underlies the difference between a modern-day smart phone and a
simple pocket calculator: a comparable example from biology would
be the difference between a cell and an individual macromolecular
complex (for example, a ribosome). Here we outline two approaches
where an investment of resources and effort may sustain similar exponential growth in the complexity of DNA nanostructures over the
NATURE NANOTECHNOLOGY | VOL 6 | DECEMBER 2011 | www.nature.com/naturenanotechnology
© 2011 Macmillan Publishers Limited. All rights reserved
Figure 2 | Challenges for DNA nanostructures. a, Expanding size and complexity. Two main approaches are being explored to overcome the current
dependence of the structural DNA nanotechnology community on the viral M13 genome: the use of longer DNA scaffold strands (top left) to fold
larger structures (top right), or the assembly of pre-formed structures for the constructions of supramolecular assemblies (bottom). b, New functional
nanostructures. The functionalization of specific protein surface residues (dark blue circles on the light blue proteins) with oligonucleotides, and
subsequent purification, would allow for an extra dimension of positioning control of the protein into a DNA template. c, New generation of DNA walkers
(green spheres with purple legs) with programmable routines and/or sensitive to state changes, such as light, for the selection of routes in multi-path
systems. d, In vivo selection and amplification of DNA nanostructures. Creating procedures for the selection and evolution of biocompatible/bioactive
shapes through environmental conditioning, or using cellular replication machinery for the high-throughput production of DNA structures, should lead to
new applications of DNA nanotechnology.
next two decades. Two of the most prominent obstacles are the high
cost of synthetic DNA and the high error rate of self-assembly.
DNA synthesis and sequence design. At current prices of about
US$0.10 per base for oligonucleotide synthesis on the 25-nmol scale,
the overall material cost for constructing a new M13-based origami
is around US$700. A key technological opportunity is the emerging
commercial availability of affordable arrays on which small amounts
of each of the tens of thousands of unique oligonucleotide sequences
are printed at a current price of less than US$0.001 per base. If reliable low-cost methods for enzymatic amplification of subsets of
strands from these arrays could be further developed81,82, this would
raise the possibility of custom-designed DNA nanostructures that are
1 gigadalton in mass (that is, around 100 times as complex as current
M13-based origami) for a material cost of ~US$1,000. Large reductions in cost of enzymatic amplification would also enable production of gram to kilogram quantities of complex DNA nanostructures,
which will be important for many but not all applications.
For DNA origami, a current constraint has been the reliance
on the 7 kb genome of M13 as the primary source of scaffold.
To create larger structures, ideally one could fold either a longer
unique scaffold, or as an alternative, multiple scaffolds with distinct sequences. Furthermore, it seems unlikely that M13 encodes
the optimal sequence for high-yield folding of all possible DNA
nanostructures. Thus, one would want to generate many unique
scaffold sequences, each tailored for optimized folding into a particular origami shape, or at least a large number of distinct generic
scaffold sequences that can support independent foldings in a single pot. Affordability is a concern, but gene synthesis from arrayprinted DNA again may provide a solution81,82. Consideration of
these issues naturally leads to the question of what the rules are for
effective sequence design, and our current ignorance in this area
warrants much future work in this direction, involving an interplay
between theory and experiment.
Hierarchical and templated assembly. Conventional DNA origami uses a single long scaffold molecule as half the material. Using
this approach to build a gigadalton DNA nanostructure, one would
need a scaffold over 1 megabase long, approaching the length of the
Escherichia coli genome (Fig. 2a, top). Such large DNA molecules are
NATURE NANOTECHNOLOGY | VOL 6 | DECEMBER 2011 | www.nature.com/naturenanotechnology
© 2011 Macmillan Publishers Limited. All rights reserved
mechanically fragile and difficult to synthesize. Instead, we can imagine current origami as ‘super-tiles’ that can be linked together hierarchically to form larger superstructures83,84 (Fig. 2a, bottom). Each
super-tile can be made a larger size by changing the design to enable
use of a higher ratio of non-scaffold to scaffold-strand mass85. The
design of super-tile interfaces will need to be optimized to improve
yield86–88. Higher-order superstructure can be further enforced by use
of a super-scaffold that organizes super-tiles89. Both a super-scaffold
and algorithmic assembly could be used to organize multiple orthogonal super-tiles in specific patterns within a given larger structure.
Also, lithographically etched surfaces could be used to template longrange order on a collection of super-tiles90–93; merging top-down with
bottom-up approaches in this way will attract the attention of the
semiconductor industry for microfabrication applications.
Finer structure control. Long-term progress towards building
large nanostructures will require a mature understanding of the
kinetics and thermodynamics of self-assembly within and between
DNA building-blocks94–96. One particular area of weakness has been
the lack of quantitative tools for analysing defect occurrence in
complex DNA nanostructures. Test structures should be designed
that sum or magnify the effect of cumulative small folding errors
to produce substantially deviated geometries that are easy to assay
using molecular imaging or other higher-throughput methods.
In addition, more work is needed to investigate kinetic aspects of
assembly, such as the order of association of staple strands to the
scaffold in DNA origami.
In addition to building larger and more complex DNA nanostructures, and reducing assembly errors, it is equally important to achieve
structural control at the finest possible level in all three dimensions.
A lattice-constrained DNA nanostructure is limited in precision to
the nanometre scale. However, just as external peptide loops can finetune the structure of an antibody or triosephosphate isomerase-barrel
protein scaffold, so we can use external forces to tweak the fine structure of a DNA nanoshape. Furthermore, lattice-constrained paradigms can be abandoned altogether at local ‘active sites’. Instead, one
can substitute binders and catalysts derived from other molecules (for
example, single-stranded DNA, single-stranded RNA or protein), in
some cases enhanced by new chemical functionalizations.
Figure 3 | DNA nanotechnology for biophysical studies. a, DNA origami
can act as fully addressable molecular pegboards that can be used as
molecular rulers for the organization of heteroelements (blue and red
spheres). The purple and green blocks can be any DNA structure that
directs the sphere position along a platform. A particularly interesting
application is the spatial arrangement of enzyme components of
cascade reactions. The relative positions of components can be designed
with nanometre accuracy, possibly allowing biochemists to suppress
diffusion-dependent effects in cascade reactions. This would open classic
biochemical systems to new functional properties, and potential improved
performances, distinct from bulk reaction measurements. Moreover, such
assemblies could be used as models of intracellular compartmentalization
or in vivo clustering. b, When current real-time measurement tools are
employed, many in vivo interactions elude detection. Fluorescence, and
in particular Förster resonance energy transfer, or single-dye fluorescent
markers, yield narrow snapshots of in vivo reality. DNA scissors, tweezers
or tensegrity structures (shown as cross-like structures within translucent
pink oval, which represents a cell) may be used for real-time and dynamic
measurement of target protein activity, or the specific detection and size
estimation of protein complexes required for cellular functions. The DNA
nanostructure switches conformation to accompany changes in the shape
and size of target structures in their native medium: this allows them to
serve as relays between the length scales associated with interactions
between protein constructs such as DNA-promoter complexes (~10s of
nanometres) and those associated with fluorescence reporting (a few
nanometres or less). Two such structures are shown here.
Precision positioning of heteroelements for functionality. The
ability to construct sophisticated machines and actuators is one of
the key technical goals of nanotechnology. Although self-assembly
of nucleic acids alone provides a rich capacity for driving active or
functional behaviour, the introduction of heteroelements such as
nanoparticles and proteins can lift DNA nanotechnology into a new
dimension of functional potential. One main challenge continues
to be efficient integration of heteroelements into DNA structures,
especially when precise control over orientation and position of the
guests is demanded. On the DNA side, the most popular starting
point has been commercially available amino- or thiol-modified
oligonucleotides, which then can be converted to different functionalities by reaction with appropriate heterobifunctional crosslinkers.
For integration of proteins, a challenge has been the coupling of
oligonucleotides to unique positions on the protein, and the subsequent purification of the conjugate97 (Fig. 2b). To accommodate the
diversity of guest proteins of interest, a suite of effective methods
will probably need to be developed. Increasingly exact control over
protein orientation could be achieved by multiple attachment to a
DNA nanostructure, or by 3D cavities that use steric interactions to
constrain the guest. If this challenge could be addressed, the active
site of enzymes may, for example, reliably be programmed to face
the exit of a molecular cavity or tubular structure.
Integration of inorganic nanoparticles into nanostructures
has also received considerable attention. Metallic nanoparticles
(in particular gold and silver) have led the way owing to their
NATURE NANOTECHNOLOGY | VOL 6 | DECEMBER 2011 | www.nature.com/naturenanotechnology
© 2011 Macmillan Publishers Limited. All rights reserved
Figure 4 | DNA nanostructures as biomimetic and in vivo active systems. Aldaye and co-workers recently reported the assembly of two enzymes of a
hydrogen-production cascade reaction using RNA arrays, which led to improved yields122. In vivo replication of complex DNA structures allows intracellular
components (blue, pink and yellow objects) to be organized with tighter and more complex spatial control for the study of cellular properties or new
capabilities due to the cytosol clustering effect. Conversely, DNA structures can be designed and ‘ …
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