Bench-Top Fabrication of Single-Molecule Nanoarrays by DNA Origami Placement

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 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 singlemolecule 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 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-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.

Engineering Inorganic Materials with DNA Nanostructures

“… 76 , 77 Nevertheless, lithography for precision placement may be remarkably simplified by a recently developed cleanroom-free benchtop technique for straightforward substrate-processing through the self-assembled colloidal NP monolayer. 93 Another feasible route to macroscopic wafer-scale assembly is to scale up the facile surface- and cation-assisted formation of DNA origami lattices. 94 This strategy could serve as an alternative to lattices used in the “DNA molecular epitaxy” approach, 75 where the repeatable units were formed through stitching together unique sets of 32 nt DNA bricks.…”

“…In substrate nanopatterning, one rather obvious obstacle arises when highly ordered DNA masks for potential downstream applications are required. For example, the current version of DNA-based lithography for metal nanostructures is demonstrated only with randomly deposited DNA nanostructures, and the DNA origami-templated optical nanocavities require predeposition lithography to allow their fine-tuned organization. , Nevertheless, lithography for precision placement may be remarkably simplified by a recently developed cleanroom-free benchtop technique for straightforward substrate-processing through the self-assembled colloidal NP monolayer . Another feasible route to macroscopic wafer-scale assembly is to scale up the facile surface- and cation-assisted formation of DNA origami lattices .…”

DNA Origami Meets Bottom-Up Nanopatterning

“…These powerful methods for precise DNA origami placement still rely on state-of-the-art e-beam lithography and, thus, preclude the adoption of this technique for many researchers. Consequently, as described in Shetty et al, they looked for alternatives to e-beam lithography for the fabrication of patterned surfaces . They introduced a benchtop technique using self-assembled colloidal nanoparticles to create large-scale, deterministic DNA origami nanoarrays, utilizing well-studied nanosphere lithography .…”

“…One possible route to achieve this goal is to use electron-beam (e-beam) lithography to pattern surfaces into “attractive” and “unattractive” regions, which then serve to template the self-ordering of nanoscopic objects. , This approach has been employed by Gopinath, Rothemund, and colleagues to arrange DNA origami − sheets into large arrays to tune nanocavity emission intensities and to control the orientation of individual dye molecules over a scale of 100 μm . Gopinath and co-workers have also employed colloidal lithography, a bottom-up approach to surface patterning, to arrange DNA origami discs on glass substrates over extended areas . With a simple protocol that can quickly be adopted by a standard biochemistry laboratory, it is now possible to position molecular breadboards deterministically on the macroscopic scale.…”

Programmed Assembly of DNA Templates by Silver Nanowires

“…Moreover, the programmed assembly of DNA was used to construct arbitrary shapes and patterns which intern utilized as higher order template for hierarchical organization of metallized nanowires, nanoarrays and biomaterials. [23][24][25][26] Until now, it is not difficult to place homogenous materials on a single DNA nanostructure with sub-10 nm precision, given that site-specific modification on the DNA nano-breadboard can be achieved by sequencespecific self-assembly. [27][28][29] For example, Fan and coworkers reported the integration of single wall-carbon nanotube (SWCNT) on 2D DO with absolute precision by modifying the ends of SWCNT with complementary oligonucleotide strands.…”