DNA Origami Characterized via Solid-State Nanopore: Insights into Nanostructure Dimensions, Rigidity and Yield

19 April 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Due to their programmability via specific base pairing, self-assembled DNA origami structures have proven to be useful for a wide variety of applications, including diagnostics, molecular computation, drug delivery, and therapeutics. Measuring and characterizing these structures is therefore of great interest and an important part of quality control. Here, we show the extent to which DNA nanostructures can be characterized by a solid-state nanopore; a non-destructive, label-free, single-molecule sensor capable of electrically detecting and characterizing charged biomolecules. We demonstrate that in addition to geometrical dimensions, nanopore sensing can provide information on the mechanical properties, assembly yield, and stability of DNA nanostructures. For this work, we use a model structure consisting of a 3 helix-bundle (3HB), i.e. three interconnected DNA double helices using a M13 scaffold folded twice on itself by short DNA staple strands, and translocate it through solid-state nanopores fabricated by controlled breakdown. We present detailed analysis of the passage characteristics of 3HB structures through nanopores under different experimental conditions which suggest that segments of locally higher flexibility are present along the nanostructure contour that allow for the otherwise rigid 3HB to fold inside nanopores. By characterizing partially melted 3HB structures, we find that locally flexible segments are likely due to short staple oligomers missing from the fully assembled structure. The 3HB used herein is a prototypical example to establish nanopores as a sensitive, non-destructive, and label-free alternative to conventional techniques such as gel electrophoresis with which to characterize DNA nanostructures.


Single-Molecule Biophysics
DNA Nanotechnology

Supplementary materials

Supplementary Information
Supplementary Information on the following topics: S1. M13mp18 Scaffold Preparation and Characterization
S2. 3HB Nanostructure Design and Sequence
 S3. Dependence of Translocations on Voltage and Salt-Concentration S4. Estimates of Nanostructure Volume – ECD Comparisons
 S5. Strong Correlation of Metastable State and Total Duration
 S6. Voltage Dependence of Metastable State Duration Distributions S7. Metastable State Power Spectra
 S8. Metastable State in Folded and Single-File Translocations
 S9. Dependence of Metastable State on Experimental Conditions S10. Gel Electrophoresis for Free-Solution Mobility Extraction
 S11. Thermal Degradation Experiments


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