DNA-BASED NANOMACHINE DESIGN

Creating nanoscale machine elements by adapting an engineering machine design approach to DNA origami

Design Concept

The concept behind our design approach is to construct stiff components (i.e. links) using bundles of double-stranded DNA and connect those links at joints formed by flexible single-stranded DNA connections. Using this idea we have fabricated hinges (1D rotational motion), sliders (1D linear motion), universal joints (2D rotational motion), and compliant joints allowing for tunable flexibility. Similar to macroscopic engineering, we built higher-order mechanisms with specific motion by integrating several links and joints into one system. The figure below shows examples of these mechanisms, which include a slider-crank (coupling rotational and linear motion), a Bennett 4-bar linkage (3D motion), a bi-stable mechanism, and a scissor mechanism.

A series of static TEM snapshots demonstrating the rotational motion of unconstrained DNA origami hinges

DNA origami mechanisms

(a) Slider crank coupling rotational and linear motion (b) Bennett 4-bar linkage moving between an expanded frame configuration and a compacted bundle (c) Bi-stable mechanism using hinges and a compliant joint to cross a designed energy barrier to switch between two stable states (d) Scissor mechanism translated rotational motion to linear motion. This structure can be polymerized to amplify extension. Scale bars are 50 nm except (b) is 100 nm. Adapted from ref. 1.

Controlling Motion of DNA Nanomachines

Rotational Control: Here, we develop methods for controlling the motion of DNA origami mechanisms, with a focus on optimizing speed of actuation. The ability to actuate dynamic systems in real time is crucial to apply them as functional devices, such as manipulators or delivery vehicles.

To demonstrate actuation of our Bennett linkage, we modified the links to include ssDNA overhangs distributed across their length. Closing DNA strands (shown in green) bind to overhangs on opposite arms, thereby pulling the structure closed. Once in the closed state, additional strands (orange) with a higher binding affinity to the closing strands can remove the closing strands and release the mechanism back to the free state. A bulk fluorescence-quenching assay was used to measure the kinetics of these reactions. Compacting and expanding occurs on the timescale of t1/2 ≈ 1 min.

Distributed actuation of DNA Origami Bennett linkage

(a) ssDNA overhangs distributed across the length of mechanism arms come together when closing strands are added. Once closed, additional releasing strands can be added to remove the closing strands via DNA strand displacement, releasing the mechanism back into the free state. This process was verified through TEM (b-d). The actuation timescales measured using a fluorescence-quenching assay reveal a compacting timescale of t1/2,c = 55 s (e) and expanding timescale of t1/2,e = 49 s (f). Scale bars = 100 nm. Figure from reference 1.

DNA-based Nanosensors

DNA nanostructures have a well-defined structure not found other soft materials, making it possible to build molecular switches that react to mechanical and chemical changes to their environment. By controlling DNA sequence, spatial design, and chemical functionalization, molecular switches can react to a library of targets including force, pH, complex ionic conditions, temperature, and the presence of specific genes or biomolecules.

Rapid actuation triggered by salt concentration

We modified a DNA origami hinge with short, weakly complementary single-stranded DNA overhangs, whose hybridization is sensitive to cation concentrations in solution. We triggered conformational changes with several different types of ions including mono-, di-, and trivalent ions and also illustrated the ability to engineer the actuation response with design parameters such as number and length of DNA overhangs and hinge torsional stiffness. Single-molecule Förster resonance energy-transfer (smFRET) measurements revealed that closing and opening transitions occur on the millisecond time scale, and these transitions can be repeated with time resolution on the scale of one second. Our results advance capabilities for rapid control of DNA nanodevices, expand the range of triggering mechanisms, and demonstrate DNA nanomachines with tunable analog responses to the local environment.

Cation-Activated Avidity for Rapid Reconfiguration of DNA Nanodevices.

Hinges can be programmed to open or close triggered by a specific ionic condition. Using single molecule FRET, we observe these conformational transitions occurring on millisecond timescales. Figure from reference 6.

DNA origami toolbox. Figure from reference 3.

References

Marras, A. E.; Zhou, L.; Su, H. J.; Castro, C. E. Proceedings of the National Academy of Sciences of the United States of America. 112, (3), 713-8 (2015).
Marras, A. E.; Zhou, L.; Kolliopoulos, V.; Su, H. J.; Castro, C. E. New Journal of Physics. 18, (5), 055005 (2016).
Castro, C. E.; Su, H. J.; Marras, A. E.; Zhou, L.; Johnson, J. Nanoscale. 7, (14), 5913-21 (2015).
Zhou, L.; Marras, A. E.; Su, H. J.; Castro, C. E. ACS Nano. 8, (1), 27-34 (2014).
Zhou, L.; Marras, A. E.; Su, H. J.; Castro, C. E. Nano Letters. 15, (3), 1815-21 (2015).
Marras, A.E.; Shi, Z., Lindell, M, Patton, R., Huang, C.-M., Zhou, L., Su, H.-J., Arya, G., Castro, C.E. ACS Nano. 12, (9), 9484-9494 (2018).

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