of 35
Low-cost, bottom-up fabrication of large-scale
single-molecule nanoarrays by DNA origami placement
Rishabh M. Shetty
a,b,e,2
, Sarah R. Brady
a
, Paul W. K. Rothemund
c
, Rizal F. Hariadi
a,d,1,2
, and Ashwin
Gopinath
c,e,1,2
a
Biodesign Center for Molecular Design and Biomimetics (at the Biodesign Institute) at Arizona State University, Tempe, AZ
85287, USA.;
b
School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85287, USA.;
c
Departments of Bioengineering, Computational and Mathematical Sciences, and Computation and Neural Systems,
California Institute of Technology, Pasadena, CA, USA.;
d
Department of Physics, Arizona State University, Tempe, AZ
85287, USA.;
e
Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
1
A.G. and R.F.H. supervised this work equally.
2
To whom correspondence should be addressed. E-mail: rmshetty@mit.edu, rhariadi@asu.edu, and agopi@mit.edu
This manuscript was compiled on August 14, 2020
L
arge-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 unique advantages of microarrays 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, transforming
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 bench-top 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%, two-fold higher than the statistical limit of 37% imposed
on non-specific 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.
Introduction
Bulk 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 Reflection Fluorescence (TIRF) microscopy
5,6
, single-molecule
experiments of biomolecular kinetics, conformational fluctuations, and folding mechanisms have become
commonplace 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 diffraction-limited spot,
often leading to confounding data (
Fig.
1A
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
Page 1 of
35
.
CC-BY-NC-ND 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted August 14, 2020.
.
https://doi.org/10.1101/2020.08.14.250951
doi:
bioRxiv preprint
controlling the positions of molecules-of-interest for optimal data quality on a substrate would require
close-packing (
Fig.
1C
), ideally at the diffraction 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 positional control on experimentally relevant substrates, and the
size of most single molecules is well below the resolution of current micro-to-nanomanipulation techniques.
DNA origami
15
is regarded as a molecular breadboard and bridge between the bottom-up worlds of
biochemistry and the top-down world of lithography
16
. DNA origami nanotechnology is modular and
spatially-programmable
17–22
; an assembled origami unit being capable of carrying up to 200 individually
addressable molecules-of-interest
23–26
. In the last decade, origami nanostructures have been utilized for
a myriad of applications ranging from electronic–
27,28
and optical–devices
14,26,29,30
, to single-molecule
biophysics
9–11,31,32
, biosensing
33–35
, and nanofabrication
36–40
. Being synthesized in solution, spatial
stochasticity is intrinsically linked with the deposition of planar origami and their payload on glass
substrates for optical experiments. A 2D DNA origami nanostructure (
100 nm), however, is more than an
order of magnitude larger than other molecules, which makes it amenable to lithographic manipulation and
deterministic positioning.
Electron Beam Lithography-based DNA Origami Placement (DOP)
36–38
leverages the ability of origami
nanostructures – through their electrostatic or covalent coupling to mica, glass, silicon, and silicon nitride –
to interface biomolecular functional moieties with the outside world for visual probing. A recent application
of this method demonstrates the large-scale integration of functionalized DNA origami through placement
on
100-nm binding sites with >90% single-binding efficiency for hybrid nanodevice fabrication
36
. Such
a composite nano-to-micro-manipulation technique enables bi-level control— first, through the arbitrary
decoration of molecules with a resolution of 5 nm on origami nanostructures, and second, by positioning
the origami themselves on lithographically-patterned sites on a desired substrate. The major drawback of
lithographic techniques for origami placement is their high-cost owing to the manufacturing complexity
of top-down fabrication. The wide-scale utilization of such processes is therefore impractical for scientific
research such as biophysics, which traditionally does not use sophisticated top-down nanofabrication.
Bottom-up, self-assembly based approaches have the unique potential to provide a framework for parallel
fabrication of structures from components either too diminutive or innumerable to be handled robotically
41
.
Such processes were predicted to be a cornerstone of the field of nanotechnology during its nascent stages
42
.
Self-assembly techniques like nanosphere lithography (NSL), while limited in terms of their ability to create
arbitrary shapes, offer a variety of advantages – they are cheap, facilitate fast, parallel-processing, and a
variety of crystallization techniques exist for covering arbitrarily large surface topologies
43,44
. In NSL, a
flat, hydrophilic substrate is coated with a monodisperse colloidal suspension of spheres, and upon drying,
a hexagonal-close-packed(HCP) layer called a Colloidal Crystal Mask is formed. Attractive capillary forces
and convective nanosphere transport are the dominant factors in the self-assembly process
43
. The order
and quality of the assembled arrays are substantially affected by the rates of solvent evaporation
45,46
.
Control over the temperature and the humidity of the system on a slightly tilted substrate can yield
colloidal monolayers
47
. Methods such as spin-coating
48
, Langmuir-Blodgett deposition
49
, and controlled
evaporation
50
have all been used to assemble large-scale monolayers of colloidal suspensions.
Here, we present the application of NSL to the controlled placement of DNA origami nanostructures on
glass substrates as a framework for the fabrication of large-scale single molecule nanoarrays. This novel
method for bench-top, cleanroom-free, DNA origami placement in meso-to-macro-scale grids utilizes tunable
colloidal nanosphere masks
44,51–54
and surface chemistry. This technique is similar to previous work
55
which patterned gold nanoparticle arrays, but here we place the emphasis on maximizing single-molecule
occupancy. Another recently introduced technique of DNA origami adsorption in nanohole arrays
39
formed
using NSL performed critical process steps circuitously in a cleanroom environment and was limited to
approximately 50% single occupancy with extremely long incubation periods. In the study reported here,
we first establish the optimal binding site diameter for circular origami and subsequently characterize the
single origami binding. We report a maximum efficiency of 74%, two-fold higher than the Poisson limit of
Page 2 of
35
.
CC-BY-NC-ND 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted August 14, 2020.
.
https://doi.org/10.1101/2020.08.14.250951
doi:
bioRxiv preprint