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Low-cost, bottom-up fabrication of large-scale single-molecule nanoarrays by DNA origami placement

Shetty, Rishabh M. and Brady, Sarah R. and Rothemund, Paul W. K. and Hariadi, Rizal F. and Gopinath, Ashwin (2020) Low-cost, bottom-up fabrication of large-scale single-molecule nanoarrays by DNA origami placement. . (Unpublished)

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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 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.

Item Type:Report or Paper (Discussion Paper)
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URLURL TypeDescription Paper ItemData/Code
Shetty, Rishabh M.0000-0003-4384-4038
Rothemund, Paul W. K.0000-0002-1653-3202
Hariadi, Rizal F.0000-0001-7840-859X
Gopinath, Ashwin0000-0002-2874-9457
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. This version posted August 14, 2020. A.G. and R.F.H. supervised this work equally. 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 National Institutes of Health Director’s New Innovator Award (1DP2AI144247) to RFH and Arizona Biomedical Research Consortium (ADHS17-00007401) to RFH; Office of Naval Research (N00014-17-1-2610 and N00014-18-1-2649) to PWKR and National Science Foundation (CCF-1317694 and CMMI-1636364) to PWKR. 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. Conflict of interest: A US patent application (WO2019108954A1) has been filed based on this work.
Funding AgencyGrant Number
Arizona Biomedical Research ConsortiumADHS17-00007401
Office of Naval Research (ONR)N00014-17-1-2610
Office of Naval Research (ONR)N00014-18-1-2649
Record Number:CaltechAUTHORS:20200819-121747023
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Official Citation:Low-cost, bottom-up fabrication of large-scale single-molecule nanoarrays by DNA origami placement. Rishabh M. Shetty, Sarah R. Brady, Paul W. K. Rothemund, Rizal F. Hariadi, Ashwin Gopinath. bioRxiv 2020.08.14.250951; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:105031
Deposited By: Tony Diaz
Deposited On:19 Aug 2020 19:41
Last Modified:19 Aug 2020 19:41

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