Recycling Materials for Sustainable DNA Origami Manufacturing

Abstract

DNA origami nanotechnology has great potential in multiple fields including biomedical, biophysical, and nanofabrication applications. However, current production pipelines lead to single-use devices incorporating a small fraction of initial reactants, resulting in a wasteful manufacturing process. Here, we introduce two complementary approaches to overcome these limitations by recycling the strand components of DNA origami nanostructures (DONs). We demonstrate reprogramming entire DONs into new devices, reusing scaffold strands. We validate this approach by reprogramming DONs with complex geometries into each other, using their distinct geometries to verify successful scaffold recycling. We reprogram one DON into a dynamic structure and show both pristine and recycled structures display similar properties. Second, we demonstrate the recovery of excess staple strands postassembly and fold DONs with these recycled strands, showing these structures exhibit the expected geometry and dynamic properties. Finally, we demonstrate the combination of both approaches, successfully fabricating DONs solely from recycled DNA components.

Keywords: DNA Nanotechnology; DNA Origami; Recycling; Reprogrammable Nanomaterials; Sustainable Manufacturing; Waste Reduction.

Figure 1

Schematic of scaffold recycling through reprogramming. (A) Folding of a DNA origami structure through initial thermal annealing (ΔT_ι). (B) Reprogramming one structure into a new structure through competitive binding of target staple strands, using thermal annealing for reprogramming (ΔT_R). TEM micrographs show representative structures after reprogramming. TEM images and oxDNA models are used to highlight the different shapes and confirm the experimental results agree with simulated structures. Scale bars: 100 nm.

Figure 2

Analysis of scaffold reprogramming. (A) Imaging analysis of cyclic scaffold reprogramming of three structures and visual comparison with pristine examples through TEM imaging. (B, C) Quantitative analysis of the scaffold reprogramming nDFS device through comparison of pristine and reprogrammed TEM micrographs (B) and measured angle conformation (C). Scale bars: 100 nm.

Figure 3

Recycling through staple recovery. (A) Schematic representation of the staple recovery process through ethanol precipitation of supernatant from PEG precipitation. (B) Agarose gel electrophoresis of nDFS.B structures folded using recycled staples over three rounds. (C) Comparison of TEM-measured angle distributions of pristine (R0) and once recycled staples (R1) nDFS.B structures with nDFS.A distribution shown for comparison. nDFS.A data was previously reported in ref (42). (D) Relative FRET efficiency of nDFS.B structure with labeled latch system for both nDFS.B-R0 and nDFS.B-R1 structures. The measurement uncertainty was smaller than the data points and has been reported with the raw FRET values in Table S7.

Figure 4

Minimal waste fabrication. (A) Reprogramming folded G-Clef and NuSpring structures into each other, using excess staple strands recovered after folding from the respective structures. The colors (green and red) indicate the source of the respective DNA component. (B) Agarose gel electrophoretic analysis of pristine and reprogrammed devices using the minimal waste fabrication protocol (L = 1 kb ladder, S = p8064 scaffold, N = pristine NuSpring, GN = G-Clef reprogrammed into NuSpring, G = pristine G-Clef, NG = reprogrammed G-Clef). (C) Representative TEM images of the reprogrammed devices. Scale bars: 100 nm.