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 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 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.
Highlights
Large-scale nanoarrays of single biomolecules enable high-throughput assays while unmasking the underlying heterogeneity within ensemble populations
DNA origami nanotechnology is modular and spatially programmable;[17−22] an assembled origami unit is capable of carrying up to 200 individually addressable molecules-of-interest.[23−26] In the past 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
We have developed a cleanroom-free, DNA origami placement technique which surpasses the single-molecule binding efficiency imposed by Poisson statistics on traditional single-molecule deposition methods
Summary
Large-scale nanoarrays of single biomolecules enable high-throughput assays while unmasking the underlying heterogeneity within ensemble populations. A recent application of this method by Gopinath et al.[36] 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 Such a composite nano-to-micro-manipulation technique enables bilevel 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. Methods such as spin-coating,[48] Langmuir−Blodgett deposition,[49] and controlled evaporation[50] have been used to assemble large-scale monolayers of colloidal suspensions
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