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Published July 27, 2021 | Supplemental Material + Submitted + Published
Journal Article Open

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


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.

Additional Information

© 2021 The Authors. Published by American Chemical Society. Attribution 4.0 International (CC BY 4.0). Received: February 6, 2021; Accepted: June 14, 2021; Published: July 6, 2021. We thank M. Kennedy and E. Le for support with data collection and H. Sasaki, A. Auer, and R. Jungmann for helpful discussions on DNA-PAINT. This work was supported by a National Institutes of Health Director's New Innovator Award (1DP2AI144247, to R.F.H.); the Arizona Biomedical Research Consortium (ADHS17-00007401, to R.F.H.); the Office of Naval Research (N00014-17-1-2610 and N00014-18-1-2649, to P.W.K.R.), and the National Science Foundation (CCF-1317694 and CMMI-1636364, to P.W.K.R. and MCB-2027165, to A.G.). AFM data were collected in the lab of H. Yan at Arizona State University. SEM images were acquired at the Center for Solid State and Electronics Research at Arizona State University. Author Contributions: A.G. and R.F.H. supervised this work equally. R.M.S., R.F.H., and A.G. designed the research; R.M.S. and S.R.B. performed experiments; R.M.S., P.W.K.R., R.F.H., and A.G. contributed new reagents/analytical tools; R.M.S., R.F.H., and A.G. analyzed data; and R.M.S., R.F.H., and A.G. wrote the paper. The authors declare the following competing financial interest(s): A US patent application (WO2019108954A1) has been filed based on this work. All data supporting the findings of this study are available within the paper and its Supporting Information. All RAW data are available upon request. All sequences of the DNA origami design are included as Supporting Information File 2 and Table S2.

Attached Files

Published - acsnano.1c01150.pdf

Submitted - 2020.08.14.250951v1.full.pdf

Supplemental Material - nn1c01150_si_001.pdf

Supplemental Material - nn1c01150_si_002.zip

Supplemental Material - nn1c01150_si_003.zip


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August 20, 2023
October 20, 2023