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Bench-Top Fabrication of Single-Molecule
Nanoarrays by DNA Origami Placement
Rishabh M. Shetty,
*
Sarah R. Brady, Paul W. K. Rothemund, Rizal F. Hariadi,
*
,
and Ashwin Gopinath
*
,
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ACSNano
2021, 15, 11441
11450
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Supporting Information
ABSTRACT:
Large-scale nanoarrays of single biomolecules
enable high-throughput assays while unmasking the underlying
heterogeneity within ensemble populations. Until recently,
creating such grids which combine the advantages of micro-
arrays and single-molecule experiments (SMEs) has been
particularly challenging due to the mismatch between the size
of these molecules and the resolution of top-down fabrication
techniques. DNA origami placement (DOP) combines two
powerful techniques to address this issue: (i) DNA origami,
which provides a
100 nm self-assembled template for single-
molecule organization with 5 nm resolution and (ii) top-down
lithography, which patterns these DNA nanostructures, trans-
forming them into functional nanodevices via large-scale
integration with arbitrary substrates. Presently, this technique relies on state-of-the-art infrastructure and highly trained
personnel, making it prohibitively expensive for researchers. Here, we introduce a cleanroom-free, $1 benchtop technique to
create meso-to-macro-scale DNA origami nanoarrays using self-assembled colloidal nanoparticles, thereby circumventing the
need for top-down fabrication. We report a maximum yield of 74%, 2-fold higher than the statistical limit of 37% imposed on
non-speci
fi
c molecular loading alternatives. Furthermore, we provide a proof-of-principle for the ability of this nanoarray
platform to transform traditionally low-throughput, stochastic, single-molecule assays into high-throughput, deterministic
ones, without compromising data quality. Our approach has the potential to democratize single-molecule nanoarrays and
demonstrates their utility as a tool for biophysical assays and diagnostics.
KEYWORDS:
DNA nanotechnology, DNA origami placement, self-assembly, nanosphere lithography, single molecule experiments,
nanoarray, Poisson statistics
B
ulk measurements yield little information about the
heterogeneity prevalent at the single-molecule level.
1
,
2
The interest in gaining quantitative and mechanistic
insight into these molecular processes spurred the development
of novel biophysical and analytical single-molecule methods
over the past few decades.
2
4
Since the introduction of total
internal re
fl
ection
fl
uorescence (TIRF) microscopy,
5
,
6
single-
molecule experiments of biomolecular kinetics, conformational
fl
uctuations, and folding mechanisms have become common-
place in biophysics laboratories.
7
,
8
Classical single-molecule
experiments such as these are stochastic in nature,
7
14
with
biophysicists lacking the ability to control where individual
molecules bind on surfaces. This leads to the possibility that two
or more molecules may occupy the same di
ff
raction-limited
spot, often leading to confounding data (
Figure 1
A and B).
Reducing the concentration of molecules to overcome this issue
has the caveat of lowering experimental throughput. This
concentration versus throughput conundrum is a major
limitation of conventional single-molecule studies.
1
Maximizing
throughput while controlling the positions of molecules-of-
interest for optimal data quality on a substrate would require
close-packing (
Figure 1
C), ideally at the di
ff
raction limit of light
λ
/2NA, where
λ
is the wavelength of excitation light, and NA is
the numerical aperture of the objective lens. However, any
deterministic positioning of molecules requires precise position-
al control on experimentally relevant substrates, and the size of
most single molecules is well below the resolution of current
micro-to-nano-manipulation techniques.
Received:
February 6, 2021
Accepted:
June 14, 2021
Published:
July 6, 2021
Article
www.acsnano.org
© 2021 The Authors. Published by
American Chemical Society
11441
https://doi.org/10.1021/acsnano.1c01150
ACSNano
2021, 15, 11441
11450
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