DNA-templated self-assembly of protein and nanoparticle linear arrays.

Abstract

Self-assembling DNA tiling lattices represent a versatile system for nanoscale construction. Self-assembled DNA arrays provide an excellent template for spatially positioning other molecules with increased relative precision and programmability. Here we report an experiment using a linear array of DNA triple crossover tiles to controllably template the self-assembly of single-layer or double-layer linear arrays of streptavidin molecules and streptavidin-conjugated nanogold particles through biotin-streptavidin interaction. The organization of streptavidin and its conjugated gold nanoparticles into periodic arrays was visualized by atomic force microscopy and scanning electron microscopy.

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DNA-Templated Self-Assembly of Protein and Nanoparticle Linear Arrays
Hanying Li, Sung Ha Park, John H. Reif, Thomas H. LaBean,* and Hao Yan*
Department of Computer Science, Duke UniVersity, Durham, North Carolina 27708
Received September 4, 2003; E-mail: hy1@cs.duke.edu; thl@cs.duke.edu
Recent years have witnessed substantial advances in the use of
DNA as a smart material to construct periodically patterned
structures.1DNA also has been designed to direct the assembly of
other functional molecules by the use of appropriate attachment
chemistries.2The diversity of materials which can be chemically
attached to DNA considerably enhances the attractiveness of DNA
nanostructures for assembly of other materials. Self-assembling
DNA tiling lattices represent a versatile system for nanoscale
construction. The methodology of DNA lattice self-assembly begins
with the chemical synthesis of single-stranded DNA molecules,
which self-assemble into DNA branched motif complexes, known
as tiles.1b-1fDNA tiles can carry sticky-ends that preferentially
match the sticky-ends of other particular DNA tiles, thereby
facilitating the further assembly into lattices. Self-assembled two-
dimensional DNA tiling lattices composed of tens of thousands of
tiles have been demonstrated.1b-1fSelf-assembled DNA arrays
provide an excellent template for spatially positioning other
molecules with increased relative precision and programmability.
Here we report an experiment using a linear array of DNA triple
crossover molecules (TX) to controllably template the self-assembly
of two forms (single-layer or double-layer) of streptavidin linear
arrays through biotin-streptavidin interaction. Figure 1 illustrates
the design. The TX molecule used here was derived from the DNA
motif described elsewhere,1cand it consists of seven oligonucle-
otides hybridized to form three double-stranded helices lying in a
plane and linked by strand exchange at four immobile crossover
points. The TX molecule shown in Figure 1a is designed such that
it contains two stem loops protruding, one each out of the upper
and the lower helices. A linear array of the TX molecules can be
obtained by designing three pairs of sticky ends where their
complementarity is represented by matching color and geometric
shape (Figure 1b). To template the assembly of streptavidin
molecules, the hairpin loops were modified to incorporate two biotin
groups per loop, indicated by the small blue dots. Formation of
single-layer or double-layer streptavidin linear arrays was controlled
using two different templates which are illustrated in Figure 1b. In
the first template (left panel), only one stem loop in each TX
molecule was modified with biotin groups. However, in the second
template (right panel), both stem loops were modified to incorporate
biotins. The binding of streptavidin molecules, which is represented
as yellow dots, to the two different templates resulted in single-
layer or double-layer streptavidin linear arrays, as shown in Figure
1b.
Streptavidin has a diameter of ∼4 nm. Its binding to the self-
assembled TX array generates bumps at biotinylated locations on
hairpin loops of the TX tiles which can be detected by atomic force
microscopy imaging (AFM). Figure 2a shows an AFM image of a
sample containing only streptavidin, demonstrating that the strepta-
vidin molecules are randomly distributed on the surface. Figure 2b
shows an AFM image of the bare TX linear DNA array. The length
of each hairpin loop is ∼3.4 nm (10 base pairs) and is not resolved
due to the well-known limitation of the lateral resolution of AFM.
However, the binding of 4 nm streptavidin to each biotinylated
hairpin loop dramatically enhances its visibility by AFM. Figure
2c shows an AFM image of the TX-templated single-layer
streptavidin linear arrays, where only one side loop in each TX
tile is modified with biotin. The streptavidin molecules appeared
periodically on one side of the array. The measured distance
between each adjacent streptavidin molecules is ∼17 nm, matching
10.1021/ja0383367 CCC: $27.50 © 2004 American Chemical Society
Figure 1. Schematic drawing of the TX DNA-templated self-assembly of
streptavidin linear arrays.
Figure 2. AFM images of the DNA-templated protein arrays. (a) strepta-
vidin alone. (b) bare TX DNA tile arrays (c) single-layer streptavidin array
(d) double-layer streptavidin array.
Published on Web 12/23/2003
418 9 J. AM. CHEM. SOC. 2004, 126, 418-419

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the designed distance between each two adjacent repeating hairpin
loops along the linear TX arrays. AFM height measurements (see
Supporting Information) show that the streptavidin has a height of
∼3.8 nm, compared to the height of ∼1.8 nm measured on bare
DNA. This further confirms that the periodic bumps in the linear
array resulted from the binding of streptavidin molecules. Double-
layer streptavidin linear arrays were also obtained by replacing only
one strand in the first template to incorporate biotin groups on both
hairpin loops. The AFM image in Figure 2d shows the formation
of the double-layer protein linear array illustrated in Figure 1b,
demonstrating the programmability of DNA nanostructures for
templating of protein arrays. Since streptavidin is tetravalent
(capable of binding four biotin molecules), the relative concentra-
tions of DNA assembly and streptavidin protein were adjusted to
ensure each array contained only a single layer of DNA lattice. At
lower relative streptavidin concentrations, cross-linked superstruc-
tures containing one protein layer sandwiched between two DNA
layers were observed (see Supporting Information).
One potential application of DNA nanotechnology is the use of
self-assembled DNA lattices to scaffold assembly of nanoelectronic
components, especially metallic nanoparticles. Here we demonstrate
the use of the linear TX arrays for the assembly of streptavidin-
conjugated 5-nm gold particles, where the gold can be precisely
positioned periodically on the self-assembled DNA array. Panels c
and d of Figure 3 are scanning electron microscopy (SEM) images
showing the TX array templated self-assembly of single-layer and
double-layer streptavidin-gold arrays. These assemblies can be
compared to the SEM images of randomly distributed streptavidin-
conjugated gold nanoparticles (Figure 3a) and bare TX DNA
assemblies (Figure 3b). The distance between adjacent gold particles
in the single-layer and double-layer arrays is ∼17 nm, matching
the designed structures. We note that the average length of observed
linear arrays incorporating gold nanoparticles is decreased compared
to the average length of streptavidin arrays without gold. Possible
explanations for this trend include repulsion between neighboring
gold nanoparticles carrying electrical charges, decreased biotin-
streptavidin binding affinity following gold conjugation, or steric
constraints due to the additional mass of the attached gold. Further
experiments are required to differentiate between these mechanisms.
In summary, we were able to use self-assembled DNA nano-
structures to precisely control the spatial location of both strepta-
vidin molecules and their nanogold conjugates. The specific biotin-
streptavidin interaction, combined with the programmability of
DNA nanostructures, may lead to more complex patterned structures
with addressable features. The organization of streptavidin-
conjugated gold nanoparticles into periodic arrays templated by
DNA nanostructures provides a convenient way to construct
multiple nanoparticle arrays for electrical measurements3by
increasing the size of the nanogold. It may also find applications
in constructing logical molecular electronic devices such as quantum
cellular automata,4or serve as interconnects between other nano-
electronic and molecular electronic components by providing
uniformly sized gaps between adjacent gold nanoparticles.
Acknowledgment. This work has been supported by grants from
NSF to H.Y., T.H.L., and J.H.R. (EIA-00-86015, EIA-0218376,
EIA-0218359) and DARPA/AFSOR to J.H.R. (F30602-01-2-0561).
Supporting Information Available: Materials and methods, details
of DNA sequences, AFM height measurements, AFM images of cross-
linked DNA-streptavidin arrays, additional SEM images (PDF). This
material is available free of charge via the Internet at http://pubs.acs.org.
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JA0383367
Figure 3. SEM images of the DNA-templated streptavidin-nanogold
arrays, scale bars: 50 nm. (a) streptavidin-gold alone. (b) bare TX DNA
arrays (c) single-layer streptavidin-gold arrays (d) double-layer streptavi-
din-gold arrays.
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